WO2016128060A1 - Predicting t cell epitopes useful for vaccination - Google Patents

Predicting t cell epitopes useful for vaccination Download PDF

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Publication number
WO2016128060A1
WO2016128060A1 PCT/EP2015/053021 EP2015053021W WO2016128060A1 WO 2016128060 A1 WO2016128060 A1 WO 2016128060A1 EP 2015053021 W EP2015053021 W EP 2015053021W WO 2016128060 A1 WO2016128060 A1 WO 2016128060A1
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Prior art keywords
cells
protein
tumor
modified
rna
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PCT/EP2015/053021
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English (en)
French (fr)
Inventor
Ugur Sahin
Martin LÖWER
Arbel D. Tadmor
Sebastian BOEGEL
Barbara SCHRÖRS
Mathias VORMEHR
Sebastian Kreiter
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Biontech Ag
Tron - Translationale Onkologie An Der Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Ggmbh
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Priority to PCT/EP2015/053021 priority Critical patent/WO2016128060A1/en
Priority to KR1020177025724A priority patent/KR102359213B1/ko
Priority to CA2982971A priority patent/CA2982971A1/en
Priority to AU2016217965A priority patent/AU2016217965B2/en
Priority to BR112017017293A priority patent/BR112017017293A2/pt
Priority to EP21191307.4A priority patent/EP3954383A1/en
Priority to EP16704567.3A priority patent/EP3256853B1/en
Priority to JP2017542106A priority patent/JP6882178B2/ja
Priority to PCT/EP2016/052684 priority patent/WO2016128376A1/en
Priority to KR1020247017236A priority patent/KR20240095527A/ko
Priority to KR1020227003525A priority patent/KR102670064B1/ko
Priority to SI201631328T priority patent/SI3256853T1/sl
Priority to CN201680010134.9A priority patent/CN107430132B/zh
Priority to LTEPPCT/EP2016/052684T priority patent/LT3256853T/lt
Priority to PL16704567T priority patent/PL3256853T3/pl
Priority to DK16704567.3T priority patent/DK3256853T3/da
Priority to RS20211140A priority patent/RS62416B1/sr
Priority to US15/550,286 priority patent/US11156617B2/en
Priority to PT167045673T priority patent/PT3256853T/pt
Priority to HUE16704567A priority patent/HUE056658T2/hu
Priority to ES16704567T priority patent/ES2888430T3/es
Priority to CN202110651565.6A priority patent/CN113484523A/zh
Priority to MX2017010332A priority patent/MX2017010332A/es
Publication of WO2016128060A1 publication Critical patent/WO2016128060A1/en
Priority to IL253627A priority patent/IL253627B/en
Priority to MX2023002858A priority patent/MX2023002858A/es
Priority to MX2022000012A priority patent/MX2022000012A/es
Priority to HK18106129.1A priority patent/HK1246852A1/zh
Priority to JP2021078678A priority patent/JP7244571B2/ja
Priority to HRP20211419TT priority patent/HRP20211419T1/hr
Priority to CY20211100811T priority patent/CY1124558T1/el
Priority to US17/481,243 priority patent/US20220074948A1/en
Priority to AU2022215196A priority patent/AU2022215196A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to methods for predicting T cell epitopes useful for vaccination.
  • the present invention relates to methods for predicting whether modifications in peptides or polypeptides such as tumor- associ ated neoantigens are immunogenic and, in particular, useful for vaccination, or for predicting which of such modifications are most immunogenic and, in particular, most useful for vaccination.
  • the methods of the invention may be used, in particular, for the provision of vaccines which are specific for a patient's tumor and, thus, in the context of personalized cancer vaccines.
  • the present invention relates to a method for predicting immunogenic amino acid modifications, the method comprising the steps:
  • a score for binding to one or more MHC class II molecules indicating binding to one or more MHC class II molecules and a score for expression or abundance of the modified protein indicating expression, high level of expression or abundance of the modified protein indicates that the modification or modified peptide is immunogenic.
  • the present invention relates to a method for selecting and/or ranking immunogenic amino acid modifications, the method comprising the steps: a) ascertaining a score for binding of a modified peptide which is a fragment of a modified protein to one or more MHC class II molecules,
  • the method comprises performing steps a) and b) on two or more different modifications.
  • the different modifications are present in the same and/or in different proteins.
  • the method comprises comparing the scores of said two or more different modifications.
  • the scores of said two or more different modifications arc compared by ranking the different modifications by their MHC class II binding scores and removing modifications with an expression or abundance of less than a given threshold.
  • the score for binding to one or more MHC class II molecules reflects a probability for binding to one or more MHC class II molecules. In one embodiment, the score for binding to one or more MHC class II molecules is ascertained by a process comprising a sequence comparison with a database of MHC class II-binding motifs.
  • the method comprises performing step a) on two or more different modified peptides, said two or more different modified peptides comprising the same modification(s).
  • the two or more different modified peptides comprising the same modification(s) comprise different fragments of a modified protein, said different fragments comprising the same modification(s) present in the protein.
  • the two or more different modified peptides comprising the same modification(s) comprise different potential MHC class II binding fragments of a modified protein, said fragments comprising the same modification(s) present in the protein.
  • the method further comprises selecting (the) modified peptide(s) from the two or more different modified peptides comprising the same modification(s) having a probability or having the highest probability for binding to one or more MHC class II molecules.
  • the two or more different modified peptides comprising the same modification(s) differ in length and/or position of the modification(s).
  • the best score for binding to one or more MHC class II molecules of the two or more different modified peptides comprising the same modification(s) is assigned to the modification(s).
  • ascertaining a score for expression or abundance of a modified protein comprises determining the level of expression of the protein to which the modification is associated and determining the frequency of the modified protein among the protein to which the modification is associated. In one embodiment, said determining the level of expression of the protein to which the modification is associated and/or determining the frequency of the modified protein among the protein to which the modification is associated is performed on the RNA level. In one embodiment, the frequency of the modified protein among the protein to which the modification is associated is determined by determining the variant allele frequency. In one embodiment, the variant allele frequency is the sum of detected sequences, in particular reads, covering the mutation site and carrying the mutation divided by the sum of all detected sequences, in particular reads, covering the mutation site.
  • the variant allele frequency is the sum of mutated nucleotides at the mutation site divided by the sum of all nucleotides determined at the mutation site.
  • for ascertaining a score for expression or abundance of a modified protein a score for the level of expression of the protein to which the modification is associated is multiplied with a score for the frequency of the modified protein among the protein to which the modification is associated.
  • the modified peptide comprises a fragment of the modified protein, said fragment comprising the modification present in the protein.
  • the method further comprises identifying non- synonymous mutations in one or more protein -coding regions.
  • amino acid modifications arc identified by partially or completely sequencing the genome or transcriptome of one or more cells such as one or more cancer cells and optionally one or more non-cancerous cells and identifying mutations in one or more protein-coding regions.
  • said mutations are somatic mutations.
  • said mutations are cancer mutations.
  • the method is used in the manufacture of a vaccine.
  • the vaccine is derived from (a) modification(s) or (a) modified peptide(s) predicted as immunogenic or more immunogenic by said method.
  • the modification(s) are present in said patient and said ascertaining a score for binding to one or more MHC class II molecules, and said ascertaining a score for expression or abundance of the modified protein is performed for said patient.
  • said one or more MHC class II molecules are present in said patient (in this embodiment the present invention may include determining the partial or complete MHC class II expression pattern of the patient).
  • said ascertaining a score for expression or abundance of the modified protein is performed on a sample from said patient such as a tumor specimen.
  • the present invention relates to a method for providing a vaccine comprising the step:
  • the method further comprises the step:
  • a vaccine comprising a peptide or polypeptide comprising the modification(s) or modified peptide(s) predicted as immunogenic or more immunogenic, or a nucleic acid encoding the peptide or polypeptide.
  • the present invention provides a vaccine which is obtainable using the methods according to the invention. Preferred embodiments of such vaccines are described herein.
  • a vaccine provided according to the invention may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers etc.
  • the vaccine may in the form of a therapeutic or prophylactic vaccine.
  • Another aspect relates to a method for inducing an immune response in a patient, comprising administering to the patient a vaccine provided according to the invention.
  • Another aspect relates to a method of treating a cancer patient comprising the steps:
  • Another aspect relates to a method of treating a cancer patient comprising administering the vaccine according to the invention to the patient.
  • the invention provides the vaccines described herein for use in the methods of treatment described herein, in particular for use in treating or preventing cancer.
  • the treatments of cancer described herein can be combined with surgical resection and/or radiation and/or traditional chemotherapy.
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H. G. W. Leuenberger, B. Nagel, and H, Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.
  • peptide refers to substances comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids joined covalently by peptide bonds.
  • polypeptide or "protein” refers to large peptides, preferably to peptides with more than 100 amino acid residues, but in general the terms “peptide”, “polypeptide” and “protein” are synonyms and are used interchangeably herein.
  • the term "modification" with respect to peptides, polypeptides or proteins relates to a sequence change in a peptide, polypeptide or protein compared to a parental sequence such as the sequence of a wildtype peptide, polypeptide or protein.
  • the term includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants and amino acid substitution variants, preferably amino acid substitution variants. All these sequence changes according to the invention may potentially create new epitopes.
  • Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence.
  • Amino acid addition variants comprise amino- and/or carboxy-termi nal fusions of one or more amino acids, such as 1 , 2, 3, 4 or 5, or more amino acids.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1 , 2, 3. 4 or 5, or more amino acids.
  • Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place.
  • a modification or modified peptide used for testing in the methods of the invention may be derived from a protein comprising a modification.
  • derived means according to the invention that a particular entity, in particular a particular peptide sequence, is present in the object from which it is derived.
  • derived in particular means that the relevant amino acid sequence is derived from an amino acid sequence in which it is present.
  • a protein comprising a modification from which a modification or modified peptide used for testing in the methods of the invention may be derived may be a neoantigen.
  • the term "neoantigen” relates to a peptide or protein including one or more amino acid modifications compared to the parental peptide or protein.
  • the neoantigen may be a tumor-associated neoantigen, wherein the term “tumor-associated neoantigen” includes a peptide or protein including amino acid modifications due to tumor- specific mutations.
  • the term “tumor-specific mutation” or “cancer-specific mutation” relates to a somatic mutation that is present in the nucleic acid of a tumor or cancer cell but absent in the nucleic acid of a corresponding normal, i.e. non-tumorous or non-cancerous, cell.
  • the terms "turn or- specific mutation” and “tumor mutation” and the terms “cancer-specific mutation” and “cancer mutation” are used interchangeably herein.
  • immune response refers to an integrated bodily response to a target such as an antigen and preferably refers to a cellular immune response or a cellular as well as a humoral immune response.
  • the immune response may be protective/preventive/prophylactic and/or therapeutic.
  • “Inducing an immune response” may mean that there was no immune response before induction, but it may also mean that there was a certain level of immune response before induction and after induction said immune response is enhanced. Thus, “inducing an immune response” also includes “enhancing an immune response”.
  • said subject is protected from developing a disease such as a cancer disease or the disease condition is ameliorated by inducing an immune response.
  • an immune response against a tumor-expressed antigen may be induced in a patient having a cancer disease or in a subject being at risk of developing a cancer disease.
  • Inducing an immune response in this case may mean that the disease condition of the subject is ameliorated, that the subject does not develop metastases, or that the subject being at risk of developing a cancer disease does not develop a cancer disease.
  • the terms "cellular immune response” and “cellular response” or similar terms refer to an immune response directed to cells characterized by presentation of an antigen with class I or class II MHC involving T cells or T-lymphocytes which act as either "helpers” or “killers”.
  • the helper T cells also termed CD4 + T cells
  • the killer cells also termed cytotoxic T cells, cytolytic T cells, CDS ' T cells or CTLs kill diseased cells such as cancer cells, preventing the production of more diseased cells.
  • the present invention involves the stimulation of an anti-tumor CTL response against tumor cells expressing one or more tumor-expressed antigens and preferably presenting such tumor-expressed antigens with class I MHC.
  • an “antigen” covers any substance, preferably a peptide or protein. that is a target of and/or induces an immune response such as a specific reaction with antibodies or T-lymphocytes (T cells).
  • an antigen comprises at least one epitope such as a T cell epitope.
  • an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen (including cells expressing the antigen).
  • the antigen or a T cell epitope thereof is preferably presented by a cell, preferably by an antigen presenting cell which includes a diseased cell, in particular a cancer cell, in the context of MHC molecules, which results in an immune response against the antigen (including cells expressing the antigen).
  • an antigen is a tumor antigen (also termed tumor-expressed antigen herein), i.e., a part of a tumor cell such as a protein or peptide expressed in a tumor cell which may be derived from the cytoplasm, the cell surface or the cell nucleus, in particular those which primarily occur intracellularly or as surface antigens of tumor cells.
  • tumor antigens include the carcinoembryonal antigen, a 1 -fetoprotein, i so ferritin, and fetal sulphoglycoprotein, 2-H-ferroprotein and ⁇ -fetoprotein.
  • a tumor antigen preferably comprises any antigen which is expressed in and optionally characteristic with respect to type and/or expression level for tumors or cancers as well as for tumor or cancer cells, i.e. a tumor-associated antigen.
  • tumor-associated antigen relates to proteins that arc under normal conditions specifically expressed in a limited number of tissues and/or organs or in specific developmental stages, for example, the tumor-associated antigens may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are expressed or aberrantly expressed in one or more tumor or cancer tissues.
  • the tumor antigens in the context of the present invention include, for example, differentiation antigens, preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, canccr/tcstis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta, and germ line specific antigens.
  • differentiation antigens preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage
  • canccr/tcstis antigens i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta
  • germ line specific antigens i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta
  • the tumor antigen or the aberrant expression of the tumor antigen identifies cancer cells.
  • the tumor antigen that is expressed by a cancer cell in a subject is preferably a self-protein in said subject, in preferred embodiments, the tumor antigen in the context of the present invention is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e.. tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which arc not or only hardly accessible by the immune system.
  • tumor antigen relates to the relative effectivity to induce an immune response that is preferably associated with therapeutic treatments, such as treatments against cancers.
  • immunogenic relates to the property of having immunogenicity.
  • immunogenic modification when used in the context of a peptide, polypeptide or protein relates to the effectivity of said peptide, polypeptide or protein to induce an immune response that is caused by and/or directed against said modification.
  • the non- modified peptide, polypeptide or protein does not induce an immune response, induces a different immune response or induces a different level, preferably a lower level, of immune response.
  • the term "immunogenicity" o "immunogenic” preferably relates to the relative effectivity to induce a biologically relev ant immune response, in particular an immune response which is useful for vaccination.
  • an amino acid modification or modified peptide is immunogenic if it induces an immune response against the target modification in a subject, which immune response may be beneficial for therapeutic or prophylactic purposes.
  • MHC major histocompatibility complex
  • MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptides and present them for recognition by T cell receptors.
  • the proteins encoded by the MHC are expressed on the surface of cells, and display both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to a T cell.
  • the MHC region is divided into three subgroups, class I, class II, and class III.
  • MHC class 1 proteins contain an a-chain and p2-microglobulin (not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. On most immune system cells, specifically on antigen-presenting cells. MHC class II proteins contain a- and ⁇ -chains and they present antigen fragments to T-helper cells. MHC class III region encodes for other immune components, such as complement components and some that encode cytokines.
  • the MHC is both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).
  • haplotype refers to the HLA alleles found on one chromosome and the proteins encoded thereby. Haplotype may also refer to the allele present at any one locus within the MHC.
  • Each class of MHC is represented by several loci: e.g., HLA- A (Human Leukocyte Antigen-A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-P and I ILA-V for class I and HLA-DRA.
  • HLA-DRB 1 -9 HLA-, HLA-DQA1 , HLA- DQB1, HLA-DPA l , HLA-DPBl .
  • HLA allele and “MHC allele” are used interchangeably herein.
  • MHCs exhibit extreme polymorphism: within the human population there are, at each genetic locus, a great number of haplotypes comprising distinct alleles. Different polymorphic MHC alleles, of both class I and class II, have different peptide specificities: each allele encodes proteins that bind peptides exhibiting particular sequence patterns.
  • an MHC molecule is an HLA molecule.
  • MHC class II includes HLA-DM, HLA-DO, HLA-DP, HLA-DQ and HLA-DR.
  • MHC binding peptide includes MHC class I and/or class II binding peptides or peptides that can be processed to produce MHC class I and/or class II binding peptides.
  • the binding peptides are typically 8-12, preferably 8- 10 amino acids long although longer or shorter peptides may be effective.
  • the binding peptides are typically 9-30, preferably 10-25 amino acids long and are in particular 13- 18 amino acids long, whereas longer and shorter peptides may be effective.
  • a peptide If a peptide is to be presented directly, i.e., without processing, in particular without cleavage, it has a length which is suitable for binding to an MHC molecule, in particular a class I MHC molecule, and preferably is 7-30 amino acids in length such as 7-20 amino acids in length, more preferably 7- 12 amino acids in length, more preferably 8-1 1 amino acids in length, in particular 9 or 10 amino acids in length.
  • a peptide is part of a larger entity comprising additional sequences, e.g. of a vaccine sequence or polypeptide, and is to be presented following processing, in particular following cleavage
  • the peptide produced by processing has a length which is suitable for binding to an MHC molecule, in particular a class I MHC molecule, and preferably is 7-30 amino acids in length such as 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-1 1 amino acids in length, in particular 9 or 10 amino acids in length.
  • the sequence of the peptide which is to be presented following processing is derived from the amino acid sequence of an antigen or polypeptide used for vaccination, i.e., its sequence substantially corresponds and is preferably completely identical to a fragment of the antigen or polypeptide.
  • an MHC binding peptide in one embodiment comprises a sequence which substantially corresponds and is preferably completely identical to a fragment of an antigen.
  • epitope refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by a T cell, in particular when presented in the context of MHC molecules.
  • An epitope of a protein such as a tumor antigen preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 9, 10. 1 1 , 12, 13, 14, 15. 16, 17, 18, 1 , 20, 21 , 22, 23, 24. or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present invention is a T cell epitope.
  • an epitope may bind to MHC molecules such as MHC molecules on the surface of a cell and thus, may be a "MHC binding peptide”.
  • nco-epitope refers to an epitope that is not present in a reference such as a normal non-cancerous or germ line cell but is found in cancer cells. This includes, in particular, situations wherein in a normal non-cancerous or gennline cell a corresponding epitope is found, however, due to one or more mutations in a cancer cell the sequence of the epitope is changed so as to result in the neo-epitope.
  • T cell epitope refers to a peptide which binds to a MHC molecule in a configuration recognized by a T cell receptor. Typically, T cell epitopes are presented on the surface of an antigen-presenting cell .
  • predicting immunogenic amino acid modifications refers to a prediction whether a peptide comprising such amino acid modification will be immunogenic and thus useful as epitope, in particular T cell epitope, in vaccination.
  • a T cell epitope may be present in a vaccine as a part of a larger entity such as a vaccine sequence and/or a polypeptide comprising more than one T cell epitope.
  • the presented peptide or T cell epitope is produced following suitable processing.
  • T cell epitopes may be modified at one or more residues that are not essential for TCR recognition or for binding to MHC. Such modified T cell epitopes may be considered immunologically equivalent.
  • a T cell epitope when presented by MHC and recognized by a T cell receptor is able to induce in the presence of appropriate co-stimulatory signals, clonal expansion of the T cell carrying the T cell receptor specifically recognizing the peptide/MHC-complex.
  • a T cell epitope comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of an antigen.
  • said fragment of an antigen is an MHC class I and/or class II presented peptide.
  • a T cell epitope according to the invention preferably relates to a portion or fragment of an antigen which is capable of stimulating an immune response, preferably a cellular response against the antigen or cells characterized by expression of the antigen and preferably by presentation of the antigen such as diseased cells, in particular cancer cells.
  • a T cell epitope is capable of stimulating a cellular response against a cell characterized by presentation of an antigen with class I MHC and preferably is capable of stimulating an antigen-responsive cytotoxic T-lymphocyte (CTL).
  • CTL cytotoxic T-lymphocyte
  • Antigen processing refers to the degradation of a peptide, polypeptide or protein into procession products, which are fragments of said peptide, polypeptide or protein (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen presenting cells, to specific T cells.
  • Antigen presenting cells are cells which present peptide fragments of protein antigens in association with MHC molecules on their cell surface. Some APCs may activate antigen specific T cells.
  • Professional antigen-presenting cells are very efficient at internalizing antigen, either by phagocytosis or by receptor-mediated endocytosis, and then displaying a fragment of the antigen, bound to a class II MHC molecule, on their membrane.
  • the T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell.
  • An additional co-stimulatory signal is then produced by the antigen-presenting cell, leading to activation of the T cell.
  • the expression of co-stimulatory molecules is a defining feature of professional antigen-presenting cells.
  • dendritic cells The main types of professional antigen-presenting cells are dendritic cells, which have the broadest range of antigen presentation, and are probably the most important antigen-presenting cells, macrophages, B-cells, and certain activated epithelial cells.
  • DCs Dendritic cells
  • Dendritic cells are conveniently categorized as "immature” and “mature” cells, which can be used as a simple way to discriminate between two well characterized phenotypes.
  • Immature dendritic cells are characterized as antigen presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannosc receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e. g. CD54 and CD1 1 ) and eostimulatory molecules (e. g., CD40, CD80, CD86 and 4- 1 BB).
  • Dendritic cell maturation is referred to as the status of dendritic cell activation at which such antigen-presenting dendritic cells lead to T cell priming, while presentation by immature dendritic cells results in tolerance.
  • Dendritic cell maturation is chiefly caused by biomolecules with microbial features detected by innate receptors (bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL- 1 , IFNs). ligation of CD40 on the dendritic cell surface by CD40L, and substances released from cells undergoing stressful cell death.
  • the dendritic cells can be derived by culturing bone marrow cells in vitro with cytokines, such as granulocyte- macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • Non-professional antigen-presenting cells do not constitutively express the MHC class II proteins required for interaction with naive T cells: these are expressed only upon stimulation of the non-professional antigen-presenting cells by certain cytokines such as IFNy.
  • Antigen presenting cells can be loaded with MHC class I presented peptides by transducing the cells with nucleic acid, preferably RNA, encoding a peptide or polypeptide comprising the peptide to be presented, e.g. a nucleic acid encoding an antigen or polypeptide used for vaccination.
  • a pharmaceutical composition or vaccine comprising a nucleic acid delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447. or the gene gun approach described by Mahvi et al.. Immunology and cell Biology 75: 456-460, 1997.
  • the term "antigen presenting cell” also includes target cells.
  • Target cell shall mean a cell which is a target for an immune response such as a cellular immune response.
  • Target cells include cells that present an antigen, i.e. a peptide fragment derived from an antigen, and include any undesirable cell such as a cancer cell.
  • the target cell is a cell expressing an antigen as described herein and preferably presenting said antigen with class I MHC.
  • portion refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said structure.
  • a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%. preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% f the amino acids of said amino acid sequence.
  • the portion is a discontinuous fraction said discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure, each part being a continuous element of the structure.
  • a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably not more than 4 parts of said amino acid sequence, wherein each part preferably comprises at least 5 continuous amino acids, at least 10 continuous amino acids, preferably at least 20 continuous amino acids, preferably at least 30 continuous amino acids of the amino acid sequence.
  • a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure.
  • a portion, a part or a fragment of a structure preferably comprises one or more functional properties of said structure.
  • a portion, a part or a fragment of an epitope, peptide or protein is preferably immunologically equivalent to the epitope, peptide or protein it is derived from.
  • a "part" of a structure such as an amino acid sequence preferably comprises, preferably consists of at least 10%, at least 20%, at least 30%. at least 40%, at least 50%. at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% of the entire structure or amino acid sequence.
  • immunoreactive cell in the context of the present invention relates to a cell which exerts effector functions during an immune reaction.
  • An “immunoreactive cell” preferably is capable of binding an antigen or a cell characterized by presentation of an antigen or a peptide fragment thereof (e.g. a T cell epitope) and mediating an immune response.
  • an antigen or a peptide fragment thereof e.g. a T cell epitope
  • immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.
  • immunoreactive cells are T cells, preferably CD4 ' and/or CD8 + T cells.
  • an "immunoreactive cell” recognizes an antigen or a peptide fragment thereof with some degree of specificity, in particular if presented in the context of MHC molecules such as on the surface of antigen presenting cells or diseased cells such as cancer cells.
  • said recognition enables the cell that recognizes an antigen or a peptide fragment thereof to be responsive or reactive.
  • the cell is a helper T cell (CD4 + T cell) bearing receptors that recognize an antigen or a peptide fragment thereof in the context of MHC class II molecules such responsiveness or reactivity may involve the release of cytokines and/or the activation of CD8 + lymphocytes (CTLs) and/or B-cells.
  • CTLs CD8 + lymphocytes
  • CTL responsiveness may involve the elimination of cells presented in the context of MHC class I molecules, i.e.. cells characterized by presentation f an antigen with class I MHC, for example, via apoptosis or perforin-mediated cell lysis.
  • CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN- ⁇ and TNF-a, up- regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen expressing target cells.
  • CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.
  • Such CTL that recognize an antigen or an antigen fragment and are responsive or reactive are also termed "antigen-responsive CTL" herein.
  • antigen-responsive CTL Such CTL that recognize an antigen or an antigen fragment and are responsive or reactive are also termed "antigen-responsive CTL" herein.
  • B cell such responsiveness may involve the release of immunoglobulins.
  • T cell and "T lymphocyte” are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells.
  • T helper cells CD4+ T cells
  • CTLs cytotoxic T cells
  • T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptor (TCR).
  • TCR T cell receptor
  • the thymus is the principal organ responsible for the maturation of T cells.
  • T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface.
  • Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
  • APCs antigen presenting cells
  • Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CDS glycoprotein at their surface. These cells recognize their targets by binding to antigen associated ith MHC class I, which is present on the surface of nearly every cell of the body.
  • T cells have a T cell receptor (TCR) existing as a complex of several proteins.
  • the actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCR a and TCRji) genes and are called a- and ⁇ -TCR chains, ⁇ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct I cell receptor (TCR) on their surface.
  • TCR T cell receptor
  • ⁇ T cells gamma delta T cells
  • TCR is made up of one ⁇ - chain and one ⁇ -chain. This group of T cells is much less common (2% of total T cells) than the ⁇ T cells.
  • the first signal in activation of T cells is provided by binding of the T cell receptor to a short peptide presented by the MHC on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated.
  • the partner cell is usually an antigen presenting cell such as a professional antigen presenting cell, usually a dendritic cell in the case of na ' ive responses, although B cells and macrophages can be important APCs.
  • a molecule is capable of binding to a target if it has a significant affinity for said predetermined target and binds to said predetermined target in standard assays.
  • "Affinity” or "binding affinity” is often measured by equilibrium dissociation constant ( D ).
  • D equilibrium dissociation constant
  • Cytotoxic T lymphocytes may be generated in vivo by incorporation of an antigen or a peptide fragment thereof into antigen-presenting cells in vivo.
  • the antigen or a peptide fragment thereof may be represented as protein, as DN (e.g. within a vector) or as RNA.
  • the antigen may be processed to produce a peptide partner for the MHC molecule, while a fragment thereof may be presented without the need for further processing. The latter is the case in particular, if these can bind to MHC molecules.
  • administration to a patient by intradermal injection is possible. However, injection may also be carried out intranodally into a lymph node (Maloy et al. (2001 ), Proc Natl Acad Sci USA 95:3299-303 ).
  • the resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes which then propagate.
  • Specific activation of CD4+ or CD8+ T cells may be detected in a variety of ways.
  • Methods for detecting specific T cell activation include detecting the proliferation of T cells, the production of cytokines (e.g., lymphokines), or the generation of cytolytic activity.
  • cytokines e.g., lymphokines
  • a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells.
  • CD 8+ T cells a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity.
  • cell characterized by presentation of an antigen or “cell presenting an antigen” or similar expressions is meant a cell such as a diseased cell, e.g. a cancer cell, or an antigen presenting cell presenting the antigen it expresses or a fragment derived from said antigen, e.g. by processing of the antigen, in the context of MHC molecules, in particular MHC Class I molecules.
  • disease characterized by presentation of an antigen denotes a disease involving cells characterized by presentation of an antigen, in particular with class I MHC. Presentation of an antigen by a cell may be effected by transfecting the cell with a nucleic acid such as RNA encoding the antigen.
  • fragment of an antigen which is presented or similar expressions is meant that the fragment can be presented by MHC class I or class II, preferably MHC class I, e.g. when added directly to antigen presenting cells.
  • the fragment is a fragment which is naturally presented by cells expressing an antigen.
  • immunologically equivalent means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect such as induction of a humoral and/or cellular immune response, the strength and/or duration of the induced immune reaction, or the specificity of the induced immune reaction.
  • immunologically equivalent is preferably used with respect to the immunological effects or properties of a peptide used for immunization.
  • an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.
  • immune effector functions in the context of the present invention includes any functions mediated by components of the immune system that result, for example, in the killing of tumor cells, or in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis.
  • the immune effector functions in the context of the present invention are T cell mediated effector functions.
  • Such functions comprise in the case of a helper T cell (CD4 + T cell) the recognition of an antigen or an antigen fragment in the context of MHC class II molecules by T cell receptors, the release of cytokines and/or the activation of CD8 ⁇ lymphocytes (CTLs) and/or B-cells, and in the case of CTL the recognition of an antigen or an antigen fragment in the context of MHC class I molecules by T cell receptors, the elimination of cells presented in the context of MHC class I molecules, i.e..
  • the term "score” relates to a result, usually expressed numerically, of a test or examination. Terms such as “score better” or “score best” relate to a better result or the best result of a test or examination.
  • modified peptides are scored according to their predicted ability to bind to MHC class 11 and according to the expression or abundance of the modified proteins from which the modified peptides are derived.
  • a peptide with a predicted higher ability to bind to MHC class II is scored better than a peptide with a predicted lower ability to bind to MHC class II. Furthermore, a peptide with higher expression or abundance of the corresponding modified protein is scored better than a peptide with lower expression or abundance of the corresponding modified protein.
  • ascertaining a score for binding of a peptide to one or more MHC class II molecules includes determining the likelihood of binding of a peptide to one or more MHC class II molecules.
  • a score for binding of a peptide to one or more MHC class II molecules may be ascertained by using any peptide: MHC binding predictive tools.
  • MHC binding predictive tools For example, the immune epitope database analysis resource (lEDB-AR: http://tools.iedb.org) may be used.
  • Predictions are usually made against a set of MHC class II molecules such as a set of different MHC class II alleles such as all possible MHC class II alleles or a set or subset of MHC class II alleles found in a patient.
  • the patient has the modification(s) the immunogenicity of which is to be determined according to the invention or which are to be selected and/or ranked according to their predicted immunogenicity according to the invention.
  • the vaccine described herein is to be provided ultimately for said patient.
  • the present invention may also include determining the MHC class II expression pattern of a patient.
  • the present invention also may comprise performing the method of the invention on different peptides comprising the same modification(s) and/or different modifications.
  • different peptides comprising the same modification(s) in one embodiment relates to peptides comprising or consisting of different fragments of a modified protein, said different fragments comprising the same modification(s) present in the protein but differing in length and/or position of the modification(s). If a protein has a modification at position x, two or more fragments of said protein each comprising a different sequence window of said protein covering said position x are considered different peptides comprising the same modification(s).
  • different peptides comprising different modifications in one embodiment relates to peptides either of the same and/or differing lengths comprising different modifications of either of the same and/or different proteins. If a protein has modifications at positions x and y, two fragments of said protein each comprising a sequence window of said protein covering either position x or position y are considered different peptides comprising different modifications.
  • the present invention also may comprise breaking of protein sequences having modifications the immunogenicity of which is to be determined according to the invention or which are to be selected and/or ranked according to their predicted immunogenicity according to the invention into appropriate peptide lengths for MHC binding and ascertaining scores for binding to one or more MHC class II molecules of different modified peptides comprising the same and/or different modifications of either the same and/or different proteins.
  • Outputs may be ranked and may consist of a list of peptides and their predicted scores, indicating their likelihood of binding.
  • the step of ascertaining a score for expression or abundance of the modified protein may be performed with all different modifications, a subset thereof, e.g.
  • the results may be ranked and may consist of a list of peptides and their predicted scores, indicating their likelihood of being immunogenic. According to the invention, ascertaining a score for expression or abundance of the modified protein may be performed for a patient such as a cancer patient, for example, on a tumor specimen of a patient such as a cancer patient.
  • ascertaining a score for expression or abundance o a modified protein may comprises determining the level of expression of the protein to which the modification is associated and/or determining the level of expression of RNA encoding the protein to which the modification is associated (which again may be indicative for the level of expression of the protein to which the modification is associated) and determining the frequency of the modified protein among the protein to which the modification is associated and/or determining the frequency f RNA encoding the modified protein among the RNA encoding the protein to which the modification is associated.
  • the frequency of the modified protein among the protein to which the modification is associated and/or the frequency of RNA encoding the modified protein among the RNA encoding the protein to which the modification is associated may be considered the proportion o the modified protein within the protein to which the modification is associated and/or the proportion of RNA encoding the modified protein within the RNA encoding the protein to which the modification is associated.
  • the term "protein to which the modification is associated” relates to the protein which may comprise the modification and includes the protein in its unmodified as well as modified state.
  • level of expression may refer to an absolute or relative amount.
  • amino acid modifications the immunogenicity of which is to be determined according to the present invention or which are to be selected and/or ranked according to their predicted immunogenicity according to the invention may result from mutations in the nucleic acid of a cell. Such mutations may be identified by known sequencing techniques.
  • the mutations are cancer specific somatic mutations in a tumor specimen of a cancer patient which may be determined by identifying sequence differences between the genome, exomc and/or transcriptome of a tumor specimen and the genome, exome and/or transcriptome of a non-tumorigenous specimen.
  • a tumor specimen relates to any sample such as a bodily sample derived from a patient containing or being expected of containing tumor or cancer cells.
  • the bodily sample may be any tissue sample such as blood, a tissue sample obtained from the primary tumor or from tumor metastases or any other sample containing tumor or cancer cells.
  • a bodily sample is blood and cancer specific somatic mutations or sequence differences are determined in one or more circulating tumor cells (CTCs) contained in the blood.
  • a tumor specimen relates to one or more isolated tumor or cancer cells such as circulating tumor cells (CTCs) or a sample containing one or more isolated tumor or cancer cells such as circulating tumor cells (CTCs).
  • a non-tumorigenous specimen relates to any sample such as a bodily sample derived from a patient or another individual which preferably is of the same species as the patient, preferably a healthy individual not containing or not being expected of containing tumor or cancer cells.
  • the bodily sample may be any tissue sample such as blood or a sample from a non-tumorigcnous tissue.
  • the invention may involve the determination of the cancer mutation signature of a patient.
  • the term "cancer mutation signature" may refer to all cancer mutations present in one or more cancer cells of a patient or it may refer to only a portion of the cancer mutations present in one or more cancer cells of a patient.
  • the present invention may involve the identification of all cancer specific mutations present in one or more cancer cells of a patient or it may involve the identification of only a portion of the cancer specific mutations present in one or more cancer cells of a patient.
  • the methods of the invention provides for the identification of a number of mutations which provides a sufficient number of modifications or modified peptides to be included in the methods of the invention.
  • the mutations identified according to the present invention are non-synonymous mutations, preferably non-synonymous mutations of proteins expressed in a tumor or cancer cell.
  • cancer specific somatic mutations or sequence differences are determined in the genome, preferably the entire genome, of a tumor specimen.
  • the invention may comprise identifying the cancer mutation signature of the genome, preferably the entire genome of one or more cancer cells.
  • the step of identifying cancer specific somatic mutations in a tumor specimen of a cancer patient comprises identifying the genome-wide cancer mutation profile.
  • cancer specific somatic mutations or sequence differences are determined in the exome. preferably the entire exome, of a tumor specimen.
  • the invention may comprise identifying the cancer mutation signature of the exome, preferably the entire exome of one or more cancer cells.
  • the step of identifying cancer specific somatic mutations in a tumor specimen of a cancer patient comprises identifying the exome-wide cancer mutation profile.
  • cancer specific somatic mutations or sequence differences are determined in the transcriptome, preferably the entire transcriptome, of a tumor specimen.
  • the invention may comprise identifying the cancer mutation signature of the transcriptome, preferably the entire transcriptome of one or more cancer cells.
  • the step of identifying cancer specific somatic mutations in a tumor specimen of a cancer patient comprises identifying the transcriptome-wide cancer mutation profile.
  • the step of identifying cancer specific somatic mutations or identifying sequence differences comprises single cell sequencing of one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16. 17, 18, 19, 20 or even more cancer cells.
  • the invention may comprise identifying a cancer mutation signature of said one or more cancer cells.
  • the cancer cells are circulating tumor cells. The cancer cells such as the circulating tumor cells may be isolated prior to single cell sequencing.
  • the step of identifying cancer specific somatic mutations or identifying sequence differences involves using next generation sequencing (NGS ).
  • NGS next generation sequencing
  • the step of identifying cancer specific somatic mutations or identifying sequence differences comprises sequencing genomic DNA and/or RNA of the tumor specimen.
  • sequence information obtained from the tumor specimen is preferably compared with a reference such as sequence information obtained from sequencing nucleic acid such as DNA or RNA of normal noncancerous cells such as germline cells which may either be obtained from the patient or a different individual.
  • normal genomic germline DNA is obtained from peripheral blood mononuclear cells (PBMCs)
  • the term "genome” relates to the total amount of genetic information in the chromosomes of an organism or a cell.
  • the term “exomc” refers to part of the genome of an organism formed by exons. which are coding portions of expressed genes.
  • the exome provides the genetic blueprint used in the synthesis of proteins and other functional gene products. It is the most functionally relevant part of the genome and, therefore, it is most likely to contribute to the phenotype of an organism.
  • the exome of the human genome is estimated to comprise 1.5% of the total genome (Ng, PC et a I., PLoS Gen., 4(8): 1-15, 2008).
  • transcriptome relates to the set of all RNA molecules, including niRNA, rRNA, tRNA, and other non-coding RNA produced in one cell or a population of cells.
  • the transcriptome means the set of all RNA molecules produced in one cell, a population of cells, preferably a population of cancer cells, or all cells of a given individual at a certain time point.
  • a “nucleic acid” is according to the invention preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA.
  • Nucleic acids include according to the invention genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the invention, a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can, according to the invention, be isolated.
  • isolated nucleic acid means, according to the invention, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
  • a nucleic can be employed for introduction into. i.e. transfection of, cells, in particular, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.
  • the term “genetic material” refers to isolated nucleic acid, either DNA or RNA, a section of a double helix, a section of a chromosome, or an organism's or cell's entire genome, in particular its exome or transcriptome.
  • mutation refers to a change of or difference in the nucleic acid sequence (nucleotide substitution, addition or deletion) compared to a reference.
  • a “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore are not passed on to children. These alterations can (but do not always) cause cancer or other diseases.
  • a mutation is a non-synonymous mutation.
  • non-synonymous mutation refers to a mutation, preferably a nucleotide substitution, which does result in an amino acid change such as an amino acid substitution in the translation product.
  • mutation includes point mutations, Indels, fusions, chromothripsis and RNA edits.
  • index describes a special mutation class, defined as a mutation resulting in a colocalized insertion and deletion and a net gain or loss in nucleotides.
  • indel In coding regions of the genome, unless the length of an indel is a multiple of 3, they produce a frameshift mutation.
  • Indels can be contrasted with a point mutation; where an Indel inserts and deletes nucleotides from a sequence, a point mutation is a form of substitution that replaces one of the nucleotides.
  • Fusions can generate hybrid genes formed from two previously separate genes. It can occur as the result of a translocation, interstitial deletion, or chromosomal inversion. Often, fusion genes are oncogenes. Oncogenic fusion genes may lead to a gene product with a new or different function from the two fusion partners. Alternatively, a proto-oncogene is fused to a strong promoter, and thereby the oncogenic function is set to function by an upregulation caused by the strong promoter of the upstream fusion partner. Oncogenic fusion transcripts may also be caused by trans-splicing or read -through events. According to the invention, the term "chromothripsis" refers to a genetic phenomenon by which specific regions of the genome arc shattered and then stitched together via a single devastating event.
  • RNA edit refers to molecular processes in which the information content in an RNA molecule is altered through a chemical change in the base makeup.
  • RNA editing includes nucleoside modifications such as cytidine (C) to uridine (U) and adenosine (A) to inosine (I) deaminations. as well as non-templated nucleotide additions and insertions.
  • RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.
  • cancer mutation signature refers to a set of mutations which are present in cancer cells when compared to non-cancerous reference cells.
  • a “reference” may be used to correlate and compare the results obtained in the methods of the invention from a tumor specimen.
  • the "reference” may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a cancer disease, either obtained from a patient or one or more different individuals, preferably healthy individuals, in particular individuals of the same species.
  • a “reference” can be determined empirically by testing a sufficiently large number of normal specimens.
  • NGS Next Generation Sequencing
  • Third Generation Sequencing methods might substitute for the NGS technology in the future to speed up the sequencing step of the method.
  • NGS Next Generation Sequencing
  • the terms "Next Generation Sequencing” or “NGS” in the context of the present invention mean all novel high throughput sequencing technologies which, in contrast to the "conventional” sequencing methodology known as Sanger chemistry, read nucleic acid templates randomly in parallel along the entire genome by breaking the entire genome into small pieces.
  • NGS technologies are able to deliver nucleic acid sequence information of a whole genome, exome, transcriptome (all transcribed sequences of a genome) or methylome (all methylated sequences of a genome) in very short time periods, e.g. within 1 -2 weeks, preferably within 1-7 days or most preferably within less than 24 hours and allow, in principle, single cell sequencing approaches.
  • Multiple NGS platforms which are commercially available or which are mentioned in the literature can be used in the context of the present invention e.g. those described in detail in Zhang et al. 2011: The impact of next-generation sequencing on genomics. J. Genet Genomics 38 (3), 95-109; or in Voelkerding et al. 2009: Next generation sequencing: From basic research to diagnostics. Clinical chemistry 55, 641-658.
  • NGS tech nologies/platforms are examples of such NGS tech nologies/platforms.
  • Single-molecule sequencing technologies such as e.g. implemented in the PacBio RS system of Pacific Biosciences (Menlo Park, California) or in the H el i ScopeTM platform of
  • Helicos Biosciences (Cambridge, Massachusetts). The distinct characteristic of this technology is its ability to sequence single DNA or RNA molecules without amplification, defined as Single-Molecule Real Time (SMRT) D A sequencing.
  • SMRT Single-Molecule Real Time
  • HeliScope uses a highly sensitive fluorescence detection system to directly detect each nucleotide as it is synthesized.
  • FRET fluorescence resonance energy transfer
  • Other fluorescence-based single-molecule techniques are from U.S. Genomics (GeneEngineTM) and Genovoxx (AnyGeneTM).
  • Nano-technologies for single-molecule sequencing in which various nanostructures are used which are e.g. arranged on a chip to monitor the movement of a polymerase molecule on a single strand during replication.
  • approaches based on nano-technologies are the GridON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nano-pore sequencing (HANSTM) platforms developed by Nabsys (Providence, Rhode Island), and the proprietary ligase- based DNA sequencing platform with DNA nanoball (DNB) technology called combinatorial probe- anchor ligation (cPALTM).
  • Electron microscopy based technologies for single-molecule sequencing e.g. those developed by LightSpeed Genomics (Sunnyvale, California) and Halcyon Molecular (Redwood City, California)
  • Ion semiconductor sequencing which is based on the detection of hydrogen ions that are released during the polymerisation of D A.
  • Ion Torrent Systems San Francisco, California
  • Each well holds a different DNA template.
  • Beneath the wells is an ion-sensitive layer and beneath that a proprietary Ion sensor.
  • DNA and RNA preparations serve as starting material for NGS.
  • Such nucleic acids can be easily obtained from samples such as biological material, e.g. from fresh, flash-frozen or formalin-fixed paraffin embedded tumor tissues (FFPE) or from freshly isolated cells or from CTCs which are present in the peripheral blood of patients.
  • FFPE paraffin embedded tumor tissues
  • Normal non-mutated genomic DNA or RNA can be extracted from normal, somatic tissue, however germline cells are preferred in the context of the present invention.
  • Germline DNA or RNA may be extracted from peripheral blood mononuclear cells (PBMCs) in patients with non-hematological malignancies.
  • PBMCs peripheral blood mononuclear cells
  • the sequence of a tumor sample is determined twice, three times or more.
  • the sequence of a tumor sample is determined twice, three times or more.
  • a reference sample such as the sequence of a germ line sample and/or the sequence of a tumor sample more than once by determining at least once the sequence in genomic DNA and determining at least once the sequence in RNA of said reference sample and/or of said tumor sample. For example, by determining the variations between replicates of a reference sample such as a germ line sample the expected rate of false positive (FDR) somatic mutations as a statistical quantity can be estimated.
  • FDR false positive
  • a technical repeat of the reference sample can be used as a reference to estimate the number of false positives.
  • various quality related metrics e.g. coverage or SNP quality
  • coverage or SNP quality may be combined into a single quality score using a machine learning approach. For a given somatic variation all other variations with an exceeding quality score may be counted, which enables a ranking of all variations in a dataset.
  • RNA relates to a molecule which comprises at least one ribonucleotide residue and preferably being entirely or substantially composed of ribonucleotide residues.
  • “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2'- position of a ⁇ -D-ribofuranosyl group.
  • the term “RNA” comprises double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non- naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • RNA includes and preferably relates to "mRNA".
  • mRNA means "messenger-RNA” and relates to a "transcript” which is generated by using a DNA template and encodes a peptide or polypeptide.
  • an mRNA comprises a 5'-UTR, a protein coding region, and a 3'-UTR.
  • mRNA only possesses limited half-life in cells and in vitro.
  • mRNA may be generated by in vitro transcription from a DNA template.
  • the in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.
  • RNA may be stabilized and its translation increased by one or more modifications having a stabilizing effects and/or increasing translation efficiency of RNA.
  • modifications are described, for example, in PCT/EP2006/009448 incorporated herein by reference.
  • the RNA used according to the present invention it may be modified within the coding region, i.e. the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein, so as to increase the GC-content to increase mRNA stability and to perform a cod on optimization and. thus, enhance translation in cells.
  • modified fi cation in the context of the RNA used in the present invention includes a y modification of an RNA which is not naturally present in said RNA.
  • the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating RNA with a phosphatase.
  • the RNA according to the invention may have modified ribonucleotides in order to increase its stability and/or decrease cytotoxicity.
  • 5-methylcytiditie is substituted partially or completely, preferably completely, for cytidine.
  • pseudouridine is substituted partially or completely, preferably completely, for uridine.
  • the term "modification” relates to providing an RNA with a 5 '-cap or 5 " -cap analog.
  • the term “5 '-cap” refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position.
  • the term “conventional 5 " -cap” refers to a naturally occurring RNA 5 " -cap, preferably to the 7- methylguanosine cap (m 7 G).
  • 5"-cap includes a 5 '-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and/or enhance translation of RNA if attached thereto, preferably in vivo and/or in a cell.
  • RNA with a 5 " -cap or 5 ' -cap analog may be achieved by in vitro transcription of a DNA template in presence of said 5 " -cap or 5 " -cap analog, wherein said 5 '-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5 " -cap may be attached to the RNA post- transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
  • RNA may comprise further modifications.
  • a further modification of the RNA used in the present invention may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5 " - or 3 " -untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the exchange of the existing 3'-UTR with or the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha 1 -globin. beta-globin, preferably beta-globin. more preferably human beta-globin.
  • RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence.
  • poly(A) tail or "poly-A sequence” relates to a sequence of adenyl (A) residues which typically is located on the 3 * -end of a RNA molecule and "unmasked poly-A sequence” means that the poly-A sequence at the 3' end of an RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located at the 3' end, i.e. downstream, of the poly-A sequence.
  • a long poly-A sequence of about 120 base pairs results i an optimal transcript stability and translation efficiency of RNA.
  • the RNA used according to the present invention may be modified so as to be present in conjunction with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues.
  • the poly-A sequence has a length of approximately 120 adenosine residues.
  • the poly-A sequence can be unmasked.
  • incorporation of a 3 " -non translated region (UTR) into the 3 " -non translated region of an R A molecule can result in an enhancement in translation efficiency.
  • a synergistic effect may be achieved by incorporating two or more of such 3 " -non translated regions.
  • the 3 '-non translated regions may be autologous or heterologous to the RNA into which they are introduced.
  • the 3 " -non translated region is derived from the human ⁇ -globin gene.
  • RNA relates to the "half-life" of RNA.
  • "Half-life” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.
  • the half-life of an RNA is indicative for the stability of said RNA.
  • the half-life of RNA may influence the "duration of expression" of the RNA. It can be expected that RNA having a long half-life will be expressed for an ex tended time period.
  • RNA if according to the present invention it is desired to decrease stability and/or translation efficiency of RNA, it is possible to modify RNA so as to interfere with the function of elements as described above increasing the stability and/or translation efficiency of RNA.
  • expression is used according to the invention in its most general meaning and comprises the production of RNA and/or peptides, polypeptides or proteins, e.g. by transcription and/or translation.
  • expression or “translation” relates in particular to the production of peptides, polypeptides or proteins. It also comprises partial expression of nucleic acids. Moreover, expression can be transient or stable.
  • the term expression also includes an "aberrant expression” or "abnormal expression”.
  • "Aberrant expression” or “abnormal expression” means according to the invention that expression is altered, preferably increased, compared to a reference, e.g. a state in a subject not having a disease associated with aberrant or abnormal expression of a certain protein, e.g., a tumor antigen.
  • An increase in expression refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%, or more.
  • expression is only found in a diseased tissue, while expression in a healthy tissue is repressed.
  • the term "specifically expressed” means that a protein is essentially only expressed in a specific tissue or organ.
  • a tumor antigen specifically expressed in gastric mucosa means that said protein is primarily expressed in gastric mucosa and is not expressed in other tissues or is not expressed to a significant extent in other tissue or organ types.
  • a tumor antigen may also be specifically expressed under normal conditions in more than one tissue type or organ, such as in 2 or 3 tissue types or organs, but preferably in not more than 3 different tissue or organ types. In this case, the tumor antigen is then specifically expressed in these organs. For example, if a tumor antigen is expressed under normal conditions preferably to an approximately equal extent in lung and stomach, said tumor antigen is specifically expressed in lung and stomach.
  • the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into protein.
  • the term “transcription” comprises "in vitro transcription", wherein the term “in vitro transcription” relates to a process wherein RNA. in particular mRNA, is in vitro synthesized in a cell -free system, preferably using appropriate cell extracts.
  • cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present inventio encompassed by the term "vector".
  • the RNA used in the present invention preferably is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template.
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • translation relates to the process in the ribosomes of a cell by which a strand of messenger RNA directs the assembly of a sequence of amino acids to make a peptide, polypeptide or protein.
  • Expression control sequences or regulatory sequences which according to the invention may be linked functionally with a nucleic acid, can be homologous or heterologous with respect to the nucleic acid.
  • a coding sequence and a regulatory sequence are linked together "functionally” if they are bound together cov alently. so that the transcription or translation of the coding sequence is under the control or under the influence of the regulatory sequence, if the coding sequence is to be translated into a functional protein, with functional linkage of a regulatory sequence with the coding sequence, induction of the regulatory sequence leads to a transcription of the coding sequence, without causing a reading frame shift in the coding sequence or inability of the coding sequence to be translated into the desired protein or peptide.
  • control sequence comprises, according to the invention, promoters, ribosome-binding sequences and other control elements, which control the transcription of a nucleic acid or the translation of the derived RNA.
  • the regulatory sequences can be controlled.
  • the precise structure of regulatory sequences can vary depending on the species or depending on the cell type, but generally comprises 5 '-un transcribed and 5'- and 3 " -untranslated sequences, which are involved in the initiation of transcription or translation, such as TATA-box, capping- sequence, CAAT-sequence and the like.
  • 5'-untranscribed regulatory sequences comprise a promoter region that includes a promoter sequence for transcriptional control of the functionally bound gene.
  • Regulatory sequences can also comprise enhancer sequences or upstream activator sequences.
  • RNA to be expressed in a cell is introduced into said cell.
  • the RNA that is to be introduced into a cell is obtained by in vitro transcription of an appropriate DNA template.
  • RNA capable of expressing and “RNA encoding” are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the RNA, if present in the appropriate environment, preferably within a cell, can be expressed to produce said peptide or polypeptide.
  • RNA according to the invention is able to interact with the cellular translation machinery to provide the peptide or polypeptide it is capable of expressing.
  • nucleic acids in particular exogenous or heterologous nucleic acids, in particular RNA into a cell.
  • the cell can form part of an organ, a tissue and/or an organism.
  • administration of a nucleic acid is either achieved as naked nucleic acid or in combination with an administration reagent.
  • administration of nucleic acids is in the form of naked nucleic acids.
  • the RNA is administered in combination with stabilizing substances such as RNase inhibitors.
  • the present invention also envisions the repeated introduction of nucleic acids into cells to allow sustained expression for extended time periods.
  • Cells can be transfected with any carriers with which RNA can be associated, e.g. by forming complexes with the RNA or forming vesicles in which the RNA is enclosed or encapsulated, resulting in increased stability of the RNA compared to naked RNA.
  • Carriers useful according to the invention include, for example, lipid-containing carriers such as cationic lipids, liposomes, in particular cationic liposomes, and micelles, and nanoparticles.
  • Cationic lipids may form complexes with negatively charged nucleic acids. Any cationic lipid may be used according to the invention.
  • RNA which encodes a peptide or polypeptide into a cell, in particular into a cell present in vivo, results in expression of said peptide or polypeptide in the cell.
  • the targeting of the nucleic acids to particular cells is preferred.
  • a carrier which is applied for the administration of the nucleic acid to a cell for example, a retrovirus or a liposome
  • a molecule such as an antibody which is specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell may be incorporated into the nucleic acid carrier or may be bound thereto.
  • proteins which bind to a surface membrane protein which is associated with endocytosis may be incorporated into the liposome formulation in order to enable targeting and/or uptake.
  • proteins encompass capsid proteins of fragments thereof which are specific for a particular cell type, antibodies against proteins which are internalized, proteins which target an intracellular location etc.
  • cell or "host cell” preferably is an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material.
  • An intact cell preferably is a viable cell, i.e. a living cell capable of carrying out its normal metabolic functions.
  • cell includes according to the invention prokaryotic cells (e.g., E. coli) or cukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells. HELA cells, yeast cells, and insect cells).
  • the exogenous nucleic acid may be found inside the cell (i) freely dispersed as such, (ii) incorporated in a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA.
  • Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, and primates.
  • the cells may be derived from a large number of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells.
  • the cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte, or macrophage.
  • a cell which comprises a nucleic acid molecule preferably expresses the peptide or polypeptide encoded by the nucleic acid.
  • clonal expansion refers to a process wherein a specific entity is multiplied.
  • the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the specific lymphocyte recognizing said antigen is amplified.
  • clonal expansion leads to differentiation of the lymphocytes.
  • reducing or “inhibiting” relate to the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater. 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level.
  • inhibitor or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.
  • Terms such as “increasing”, “enhancing”, “promoting” or “prolonging” preferably relate to an increase, enhancement, promotion or prolongation by about at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 80%, preferably at least 100%. preferably at least 200% and in particular at least 300%. These terms may also relate to an increase, enhancement, promotion or prolongation from zero or a non-measurable or non-detectable level to a level of more than zero or a level which is measurable or detectable.
  • the present invention provides vaccines such as cancer vaccines designed on the basis of amino acid modifications or modified peptides predicted as being immunogenic by the methods of the present invention.
  • the term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, in particular a cellular immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell.
  • a vaccine may be used for the prevention or treatment of a disease.
  • personalized cancer vaccine or “individualized cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.
  • a vaccine provided according to the invention may comprise a peptide or polypeptide comprising one or more amino acid modifications or one or more modified peptides predicted as being immunogenic by the methods of the invention or a nucleic acid, preferably RNA, encoding said peptide or polypeptide.
  • the cancer vaccines provided according to the invention when administered to a patent provide one or more T cell epitopes suitable for stimulating, priming and/or expanding T cells specific for the patient's tumor.
  • the T cells are preferably directed against cells expressing antigens from which the T cell epitopes are derived.
  • the vaccines described herein are preferably capable of inducing or promoting a cellular response, preferably cytotoxic T cell activity, against a cancer disease characterized by presentation of one or more tumor-associated neoantigens with class I MHC. Since a vaccine provided according to the present invention will target cancer specific mutations it will be specific for the patient's tumor.
  • a vaccine provided according to the invention relates to a vaccine which when administered to a patent preferably provides one or more T cell epitopes, such as 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more and preferably up to 60, up to 55, up to 50, up to 45, up to 40, up to 35 or up to 30 T cell epitopes, incorporating amino acid modifications or modified peptides predicted as being immunogenic by the methods of the invention.
  • T cell epitopes are also termed "neo-epitopes" herein.
  • the methods of the invention may comprise the further step of determining the usability of the identified amino acid modifications or modified peptides for cancer vaccination.
  • further steps can involve one or more of the following: (i) assessing whether the modifications are located in known or predicted MHC presented epitopes, (ii) in vitro and/or in silico testing whether the modifications are located in MHC presented epitopes, e.g. testing whether the modifications are part of peptide sequences which are processed into and/or presented as MHC presented epitopes, and (iii) in vitro testing whether the envisaged modified epitopes, in particular when present in their natural sequence context, e.g.
  • flanking sequences when flanked by amino acid sequences also flanking said epitopes in the naturally occurring protein, and when expressed in antigen presenting cells are able to stimulate T cells such as T cells of the patient having the desired specificity.
  • flanking sequences each may comprise 3 or more, 5 or more, 10 or more, 1 5 or more, 20 or more and preferably up to 50, up to 45. up to 40, up to 35 or up to 30 amino acids and may flank the epitope sequence N-terminally and/or C-terminally.
  • Modified peptides determined according to the invention may be ranked for their usability as epitopes for cancer vaccination.
  • the method of the invention comprises a manual or computer-based analytical process in w hich the identified modified peptides are analyzed and selected for their usability in the respective vaccine to be provided.
  • said analytical process is a computational algorithm-based process.
  • said analytical process comprises determining and/or ranking epitopes according to a prediction of their capacity of being immunogenic.
  • neo-epitopes identified according to the invention and provided by a vaccine of the invention are preferably present in the form of a polypeptide comprising said neo-epitopes such as a polyepitopic polypeptide or a nucleic acid, in particular RNA, encoding said polypeptide.
  • the neo-epitopes may be present in the polypeptide in the form of a vaccine sequence, i.e. present in their natural sequence context, e.g. flanked by amino acid sequences also flanking said epitopes in the naturally occurring protein.
  • flanking sequences each may comprise 5 or more, 10 or more, 15 or more, 20 or more and preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids and may flank the epitope sequence N-terminally and/or C- terminally.
  • a vaccine sequence may comprise 20 or more, 25 or more, 30 or more, 35 or more, 40 or more and preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids.
  • the neo-epitopes and/or vaccine sequences are lined up in the polypeptide head-to-tail.
  • the neo-epitopes and/or vaccine sequences are spaced by linkers, in particular neutral linkers.
  • linker according to the invention relates to a peptide added between two peptide domains such as epitopes or vaccine sequences to connect said peptide domains.
  • linker sequence There is no particular limitation regarding the linker sequence. However, it is preferred that the linker sequence reduces steric hindrance between the two peptide domains, is well translated, and supports or allows processing of the epitopes. Furthermore, the linker should have no or only little immunogenic sequence elements.
  • Linkers preferably should not create non- endogenous neo-epitopes like those generated from the junction suture between adjacent neo- epitopes, which might generate unwanted immune reactions. Therefore, the polyepitopic vaccine should preferably contain linker sequences which are able to reduce the number of unwanted MHC binding junction epitopes. Hoyt et al. (EMBO J. 25(8), 1720-9, 2006) and Zhang et al. (J. Biol.
  • the linker each may comprise 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more and preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids.
  • the linker is enriched in glycine and/or serine amino acids.
  • a linker is substantially composed o the amino acids glycine and serine.
  • the linker comprises the amino acid sequence (GGS) a (GSS)b(GGG) c (SSG)d(GSG) e wherein a. b, c, d and e is independently a number selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9. 10, 1 1 , 12, 13, 14. 15, 16, 17.
  • the linker comprises a sequence as described herein including the linker sequences described in the examples such as the sequence GGSGGGGSG.
  • a polypeptide incorporating one or more neo-epitopes such as a polyepitopic polypeptide according to the present invention is administered to a patient in the form of a nucleic acid, preferably RNA such as in vitro transcribed or synthetic RNA, which may be expressed in cells of a patient such as antigen presenting cells to produce the polypeptide.
  • a nucleic acid preferably RNA such as in vitro transcribed or synthetic RNA, which may be expressed in cells of a patient such as antigen presenting cells to produce the polypeptide.
  • the present invention also envisions the administration of one or more multiepitopic polypeptides which for the purpose of the present invention are comprised by the term "polyepitopic polypeptide", preferably in the form of a nucleic acid, preferably RNA such as in vitro transcribed or synthetic RNA, which may be expressed in cells of a patient such as antigen presenting cells to produce the one or more polypeptides.
  • polyepitopic polypeptide preferably in the form of a nucleic acid, preferably RNA such as in vitro transcribed or synthetic RNA, which may be expressed in cells of a patient such as antigen presenting cells to produce the one or more polypeptides.
  • the neo-epitopes provided by the different multiepitopic polypeptides may be different or partially overlapping.
  • the polypeptide according to the invention is processed to produce the neo-epitopes identified according to the invention.
  • Administration of a vaccine provided according to the invention preferably provides MHC class Il-presented epitopes that are capable of eliciting a CD4+ helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived.
  • Administration of a vaccine provided according to the invention may also provide MHC class 1-presented epitopes that are capable of eliciting a CD8+ T cell response against cells expressing antigens from which the MHC presented epitopes are derived.
  • administration of a vaccine provided according to the invention may provide one or more neo-epitopes (including known neo-epitopes and neo-epitopes identified according to the invention) as well as one or more epitopes not containing cancer specific somatic mutations but being expressed by cancer cells and preferably inducing an immune response against cancer cells, preferably a cancer specific immune response.
  • neo-epitopes including known neo-epitopes and neo-epitopes identified according to the invention
  • administration of a vaccine provided according to the invention provides neo-epitopes that are MHC class I I-presented epitopes and/or are capable of eliciting a CD4+ helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived as well as epitopes not containing cancer-specific somatic mutations that are MHC class I-presented epitopes and/or are capable of eliciting a CD8+ T cell response against cells expressing antigens from which the MHC presented epitopes are derived.
  • the epitopes not containing cancer-specific somatic mutations are derived from a tumor antigen.
  • the neo-epitopes and epitopes not containing cancer-specific somatic mutations have a synergistic effect in the treatment of cancer.
  • a vaccine provided according to the invention is useful for polyepitopic stimulation of cytotoxic and/or helper T cell responses.
  • the vaccine provided according to the invention may be a recombinant vaccine.
  • recombinant in the context of the present invention means "made through genetic engineering".
  • a "recombinant entity” such as a recombinant polypeptide in the context of the present invention is not occurring naturally, and preferably is a result of a combination of entities such as amino acid or nucleic acid sequences which are not combined in nature.
  • a recombinant polypeptide in the context of the present invention may contain several amino acid sequences such as neo-epitopes or vaccine sequences derived from different proteins or different portions of the same protein fused together, e.g., by peptide bonds or appropriate linkers.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • Agents, compositions and methods described herein can be used to treat a subject with a disease, e.g.. a disease characterized by the presence of diseased cells expressing an antigen and presenting a fragment thereof. Particularly preferred diseases are cancer diseases. Agents, compositions and methods described herein may also be used for immunization or vaccination to prevent a disease described herein.
  • a disease e.g.. a disease characterized by the presence of diseased cells expressing an antigen and presenting a fragment thereof.
  • Particularly preferred diseases are cancer diseases.
  • Agents, compositions and methods described herein may also be used for immunization or vaccination to prevent a disease described herein.
  • the term “disease” refers to any pathological state, including cancer diseases, in particular those forms of cancer diseases described herein.
  • the term “normal” refers to the healthy state or the conditions in a healthy subject or tissue, i.e., non-pathological conditions, wherein “healthy” preferably means non-cancerous.
  • Disease involving cells expressing an antigen means according to the invention that expression of the antigen in cells of a diseased tissue or organ is detected. Expression in cells of a diseased tissue or organ may be increased compared to the state in a healthy tissue or organ. An increase refers to an increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1 00%, at least 10000% or even more. In one embodiment, expression is only found in a diseased tissue, while expression in a healthy tissue is repressed. According to the invention, diseases involving or being associated with cells expressing an antigen include cancer diseases.
  • tumor refers to an abnormal growth of cells (called neoplastic cells, tumorigenous cells or tumor cells) preferably forming a swelling or lesion.
  • tumor cell an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign, pre- m align ant or malignant.
  • Cancer is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, and do not invade or metastasize. Most cancers form a tumor but some, like leukemia, do not. Malignancy, malignant neoplasm, and malignant tumor are essentially synonymous with cancer.
  • Neoplasm is an abnormal mass of tissue as a result of neoplasia. " Neoplasia (new growth in Greek) is the abnormal proliferation of cells. The growth of the cells exceeds, and is uncoordinated with that of the normal tissues around it. The growth persists in the same excessive manner even after cessation of the stimuli. It usually causes a lump or tumor. Neoplasms may be benign, pre-m align ant or malignant.
  • “Growth of a tumor” or “tumor growth” relates to the tendency of a tumor to increase its size and/or to the tendency of tumor cells to proliferate.
  • cancer and “cancer disease” are used interchangeably with the terms “tumor” and “tumor disease”.
  • Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor. These are the histology and the location, respectively.
  • cancer comprises carcinomas, adenocarcinomas, blastomas, leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof.
  • carcinomas adenocarcinomas, blastomas, leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain
  • cancer according to the invention also comprises cancer metastases and relapse of cancer.
  • metastasis is meant the spread of cancer cells from its original site to another part of the body.
  • the formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs.
  • a new tumor i.e. a secondary tumor or metastatic tumor
  • Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential.
  • the term "metastasis” according to the invention relates to "distant metastasis" which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.
  • the cells of a secondary or metastatic tumor are like those in the original tumor. This means, for example, that, if ovarian cancer metastasizes to the liver, the secondary tumor is made up of abnormal ovarian cells, not of abnormal liver cells. The tumor in the liver is then called metastatic ovarian cancer, not liver cancer.
  • CTCs circulating tumor cells
  • CTCs circulating tumor cells
  • Circulating tumor cells are found in frequencies in the order of 1-10 CTC per niL of whole blood in patients with metastatic disease.
  • Research methods have been developed to isolate CTC.
  • Several research methods have been described in the art to isolate CTCs, e.g. techniques which use of the fact that epithelial cells commonly express the cell adhesion protein EpCAM, which is absent in normal blood cells.
  • Jmmunomagnetic bead-based capture involves treating blood specimens with antibody to EpCA that has been conjugated with magnetic particles, followed by separation of tagged cells in a magnetic field. Isolated cells are then stained with antibody to another epithelial marker, cytokeratin, as well as a common leukocyte marker CD45, so as to distinguish rare CTCs from contaminating white blood cells.
  • This robust and semi-automated approach identifies CTCs with an average yield of approximately 1 CTC/mL and a purity of 0.1% (A I lard et al., 2004: Clin Cancer Res 10, 6897-6904).
  • a second method for isolating CTCs uses a microfluidie- based CTC capture device which involves flowing whole blood through a chamber embedded with 80,000 microposts that have been rendered functional by coating with antibody to EpCAM. CTCs are then stained with secondary antibodies against either cytokeratin or tissue specific markers, such as PSA in prostate cancer or HER2 in breast cancer and are visualized by automated scanning of microposts in multiple planes along three dimensional coordinates. CTC- chips are able to identifying cytokerating-positive circulating tumor cells in patients with a median yield of 50 cells/ml and purity ranging from 1-80% (Nagrath et al., 2007: Nature 450, 1235-1239).
  • CTC Circulating Tumor Cell
  • Veridex, LLC Raritan, NJ
  • FDA U.S. Food and Drug Administration
  • a relapse or recurrence occurs when a person is affected again by a condition that affected them in the past. For example, if a patient has suffered from a tumor disease, has received a successful treatment of said disease and again develops said disease said newly developed disease may be considered as relapse or recurrence.
  • a relapse or recurrence of a tumor disease may but does not necessarily occur at the site of the original tumor disease. Thus, for example, if a patient has suffered from ovarian tumor and has received a successful treatment a relapse or recurrence may be the occurrence of an ovarian tumor or the occurrence of a tumor at a site different to ovary.
  • a relapse or recurrence of a tumor also includes situations wherein a tumor occurs at a site different to the site of the original tumor as well as at the site of the original tumor.
  • the original tumor for which the patient has received a treatment is a primary tumor and the tumor at a site different to the site of the ori inal tumor is a secondary or metastatic tumor.
  • treat is meant to administer a compound or composition as described herein to a subject in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the frequency or severity of symptoms and/or recurrences in a subject who currently has or who previously has had a disease; and/or prolong, i.e. increase the lifespan of the subject.
  • treatment of a disease includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset o a disease or the symptoms thereof.
  • being at risk is meant a subject, i.e. a patient, that is identified as having a higher than normal chance of developing a disease, in particular cancer, compared to the general population.
  • a subject who has had, or who currently has, a disease, in particular cancer is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease.
  • Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases.
  • immunotherapy relates to a treatment involving activation of a specific immune reaction.
  • terms such as “protect”, “prevent”, “prophylactic”, “preventive”, or “protective” relate to the prevention or treatment or both of the occurrence and/or the propagation of a disease in a subject and, in particular, to minimizing the chance that a subject will develop a disease or to delaying the development of a disease.
  • a person at risk for a tumor as described above, would be a candidate for therapy to prevent a tumor.
  • a prophylactic administration of an immunotherapy for example, a prophylactic administration of a vaccine of the invention, preferably protects the recipient from the development of a disease.
  • a therapeutic administration of an immunotherapy for example, a therapeutic administration of a vaccine of the invention, may lead to the inhibition of the progress/growth of the disease. This comprises the deceleration of the progress/growth of the disease, in particular a disruption of the progression of the disease, which preferably leads to elimination of the disease.
  • Immunotherapy may be performed using any of a variety of techniques, in which agents provided herein function to remove diseased cells from a patient. Such removal may take place as a result of enhancing or inducing an immune response in a patient specific for an antigen or a cell expressing an antigen.
  • immunotherapy may be active immunotherapy, in which treatment relics on the in vivo stimulation of the endogenous host immune system to react against diseased cells with the administration of immune response-modifying agents (such as polypeptides and nucleic acids as provided herein).
  • immune response-modifying agents such as polypeptides and nucleic acids as provided herein.
  • agents and compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).
  • immunotherapy or “vaccination” describes the process of treating a subject with the purpose of inducing an immune response for therapeutic or prophylactic reasons.
  • in vivo relates to the situation in a subject.
  • subject means “subject”, “individual”, “organism” or “patient” are used interchangeably and relate to vertebrates, preferably mammals.
  • mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc. as well as animals in captivity such as animals of zoos.
  • animal as used herein also includes humans.
  • subject may also include a patient, i.e., an animal, preferably a human having a disease, preferably a disease as described herein.
  • autologous transplant refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection.
  • heterologous is used to describe something consisting of multiple different elements. As an example, the transfer of one individual ' s bone marrow into a different individual constitutes a heterologous transplant.
  • a heterologous gene is a gene derived from a source other than the subject.
  • compositions for an immunization or a vaccination preferably one or more agents as described herein are administered together with one or more adjuvants for inducing an immune response or for increasing an immune response.
  • adjuvant relates to compounds which prolongs or enhances or accelerates an immune response.
  • the composition of the present invention preferably exerts its effect without addition of adjuvants.
  • the composition of the present application may contain any known adjuvant.
  • Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g.. Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), liposomes, and immune-stimulating complexes.
  • adjuvants examples include monophosphoryl-lipid-A (MPL Smith line Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21 (Smith line Beecham; WO 96/33739), QS7. QS17, QS 18. and QS-L l (So et al., 1997, Mol. Cells 7: 178- 186). incomplete Freund ' s adjuvants, complete Freund ' s adjuvants, vitamin E.
  • cytokines in a vaccination, owing to their regulatory properties on lymphocytes.
  • cytokines comprise, for example, interleukin-12 (IL- 12) which was shown to increase the protective actions o vaccines (cf. Science 268: 1432- 1434, 1995), GM-CSF and IL- 18.
  • IL- 12 interleukin-12
  • GM-CSF GM-CSF
  • IL- 18 IL- 12
  • Said compounds comprise co-stimulating molecules provided in the form of proteins or nucleic acids such as B7-1 and B7-2 (CD80 and CD86, respectively).
  • a bodily sample may be a tissue sample, including body fluids, and/or a cellular sample.
  • tissue samples may be obtained in the conventional manner such as by tissue biopsy, including punch biopsy, and by taking blood, bronchial aspirate, sputum, urine, feces or other body fluids.
  • sample also includes processed samples such as fractions or isolates of biological samples, e.g. nucleic acid or cell isolates.
  • the agents such as vaccines and compositions described herein may be administered via any conventional route, including by injection or infusion.
  • the administration may be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally.
  • administration is carried out intranodally such as by injection into a lymph node.
  • Other forms of administration envision the in vitro transfection of antigen presenting cells such as dendritic cells with nucleic acids described herein followed by administration of the antigen presenting cells.
  • an "effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease.
  • the desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition.
  • an effective amount of an agent described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered o the agents described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
  • the phannaceutical compositions described herein are preferably sterile and contain an effective amount of the therapeutically active substance to generate the desired reaction or the desired effect.
  • the phannaceutical compositions described herein are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible preparation.
  • pharmaceutically compatible refers to a nontoxic material which does not interact with the action of the active component of the phannaceutical composition. Preparations of this kind may usually contain salts, buffer substances, preservatives, carriers, supplementing immunity- enhancing substances such as adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines, saponin, GM-CSF and/or RNA and, where appropriate, other therapeutically active compounds.
  • adjuvants e.g. CpG oligonucleotides, cytokines, chemokines, saponin, GM-CSF and/or RNA and, where appropriate, other therapeutically active compounds.
  • salts When used in medicine, the salts should be pharmaceutically compatible. However, salts which are not pharmaceutically compatible may used for preparing pharmaceutically compatible salts and are included in the invention.
  • Phannacologically and pharmaceutically compatible salts of this kind comprise in a non-limiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic. succinic acids, and the like.
  • Pharmaceutically compatible salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.
  • a pharmaceutical composition described herein may comprise a pharmaceutically compatible carrier.
  • carrier refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate application.
  • pharmaceutically compatible carrier includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.
  • the components of the phannaceutical composition of the invention are usually such that no interaction occurs which substantially impairs the desired phannaceutical efficacy.
  • the pharmaceutical compositions described herein may contain suitable buffer substances such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.
  • the pharmaceutical compositions may, where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se.
  • Pharmaceutical compositions of the invention may be in the form of capsules, tablets, lozenges, solutions, suspensions, syrups, elixirs or in the form of an emulsion, for example.
  • compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient.
  • suitable carriers and solvents are Ringer solution and isotonic sodium chloride solution.
  • sterile, fixed oils are used as solution or suspension medium.
  • Non synonymous cancer-associated mutations are frequently immunogenic and pre-dominantly recognized by CD4 + T cells, a,
  • Splenocytes were restimulated ex vivo with the mutated peptide or an irrelevant control peptide and tested by IFNy Elispot (see exemplaril Fig.
  • b-c T cell responses obtained by vaccinating C57BL/6 mice with epitopes mutated in the B 16F10 tumor model.
  • Left prevalence of non-immunogenic, MHC class I or class 11 restricted mutated epitopes.
  • examples for detection and typing of mutation-specific T cells see Table 1 for data on individual epitopes
  • d prevalence of non-immunogenic, MHC class I or class II restricted mutated epitopes discovered in the CT26 model.
  • MHC restriction of immunogenic mutated epitopes prioritized based on predicted MHC class I binding and selected based on either good (0.1 -2.1) or poor (>3.9) binding scores. See Table 2 for data on individual epitopes.
  • RNA pentatopes induces T cell responses against the individual mutated epitopes and confers disease control and significant survival benefit in mouse tumor models, a, Engineering of a poly-neo-epitope RNA vaccine.
  • the RNA pentatope contains five 27mer sequences connected by gly/ser linkers inserted into the pST l -Sp-MlTD- 2hBgUTR-A120 backbone.
  • RNA pentatope vaccines with mutations selected for in silico predicted favorable MHC class II binding properties and abundant expression confer potent antitumor control a, Comparison of MHC II binding scores of immunogenic and non-immunogenic mutations (medians shown), b, Mutations with high expression levels were selected with ('ME' mutations) or without ('E' mutations) considering MHC class II binding score. See also Table 4. Ten mutations out of each category represented by two pentatopes each were used for vaccination of CT26-Luc lung tumor bearing mice.
  • Tumor growth curves (left), area under the curve (mid) and ink treated lungs (right) are shown, c, Mice (5 per group) were analyzed for T cell responses against the vaccinated pentatopes by restimulation with RNA electroporated syngeneic BMDC in an IFNy KIT Spot assay. Each dot represents the mean spot count of one mouse subtracted by an irrelevant RNA control (mean+SEM).
  • Neo-epitopes predicted to bind to the patients' HLA-DRB1 alleles (percentile rank ⁇ 10 %) are shown (S CM, skin cutaneous melanoma: COAD, colon adenocarcinoma: BRCA, breast invasive carcinoma).
  • Figure 5 Calculation of variant allele frequency (VAF).
  • the figure shows an idealized gene as a combination of exons on a piece of genomic DNA (upper part) and example read sequences aligned to this locus (lower part, in a higher zoom level).
  • the site of the mutation event ("mutation site") is shown by a dashed line (upper part) or box (lower part).
  • the mutant nucleotides are colored red, the wild type nucleotides are colored green. Also the sums of those nucleotides in the VAF formula are colored accordingly.
  • FIG. 6 Influence of the expression of mutated allele on the prediction performance of MHC Il-scores. 185 selected mutations from the murine tumor models 4T1, CT26 and B 16F 10 were tested for their antigenicity. The predictive performance of the calculated MHC ll-scores was deduced from the area under the receiver operating characteristic curve (AUC, open circle). This value was subsequently recalculated after applying different thresholds for the total mRNA expression (left panel) and the expression of the mutated allele (right panel, mRNA expression * mutated allele frequency, closed circles). The maximum AUC values are indicated. The expression of the mutated allele contributes more to the improvement of the prediction performance.
  • AUC receiver operating characteristic curve
  • FIG. 7 Comparison of receiver operating characteristic (ROC) curves with and without threshold on the expression.
  • the ROC curves indicate the performance of the antigenicity prediction for all 185 selected mutations from the murine tumor models 4T1, CT26 and B 16F 10 (dotted curves) and for those mutations, for which the mRNA expression was >6 RPKM (left panel, solid curve) or the expression of the mutated allele was >4 RPKM (right panel, solid curve).
  • the selected thresholds achieved the maximum AUC values (see Figure 6).
  • Gene expression values were determined by counting reads overlapping transcript exons and junctions, and normalizing to RPKM expression units (Reads which map per kilobase of transcript length per million mapped reads). Mutation expression was determined by normalization of mutated RNA reads to the total mapped read counts multiplied by 100 million (normalized variant read counts; NVRC).
  • Mutation selection, validation and prioritization was described previously (Castle, J. C, et al., Cancer Res 72, 1081 (2012); Castle, J. C, et al, 3MC Genomics 15, 190 (2014): Lower, M., et al., PLoS Comput Biol 8, el 002714 (2012)). Mutations to be pursued were selected based on following criteria: (i) present in the respective tumor cell line sequencing triplicates and absent in the corresponding healthy tissue sample triplicates, (ii) occur in a RefSeq transcript, and (iii) cause non-synonymous changes. Further criteria were occurrence in expressed genes of tumor cell lines (median RPKM across replicates).
  • TCGA Cancer Genome Atlas
  • RNA-Seq data (TCGA) of tumor samples with identified genomic mutations was used to define expressed nsSNVs.
  • seq2HLA was employed to identify the patients " 4-digit HLA class II (HLA-DQA 1 , HLA-DQB 1. HLA-DRB 1 ) type.
  • the IEDB consensus binding prediction (version 2.12) was used to predict MHC class II binding from a 27mer peptide and the patients HLA-DRB 1 alleles.
  • neo-eptiopes with a percentile rank below 10% were considered as binders.
  • Identified non-synonymous mutations were studied in the context of the respective 27mer amino acid epitope with the utated amino acid in the center (position 14). Either of these mutated peptides were synthesized together with control peptides (vesiculo-stomatitis virus nucleo-protein (VSV-NP52-59), gp70-AHl (gp70 423 - 4 3i) and tyrosinase- related protein 2 (Trp2iso-i 8s) by JPT Peptide Technologies GmbH .
  • VSV-NP52-59 vesiculo-stomatitis virus nucleo-protein
  • gp70-AHl gp70 423 - 4 3i
  • Trp2iso-i 8s tyrosinase- related protein 2
  • sequences encoding mutated 27m er peptides were cloned into the pST 1 -Sp-MITD-2hBgUTR-A 120 backbone (Holtkamp. S., et al., Blood 108, 4009 (2006)) featuring sequence elements for pharmacologically optimized synthetic RNA in terms of translation efficiency and MHC class I/I I processing of epitopes either as monotopes or as pentatopes fused to each other by sequences encoding 10 amino acid long glycine-serine linker in between. Linearization of these plasmid constructs, in vitro translation (IVT) of these templates and purification are described in detail elsewhere (Holtkamp, S., et al.. Blood 108, 4009 (2006)).
  • Vaccination was performed either by retro-orbital injection of 200 ⁇ (20 ⁇ g per mutation for B16F10, 40 ⁇ g per mutation for CT26) RNA complexed with cationic lipids (manuscript in preparation) or subcutaneous injection of 100 ⁇ g synthetic peptide and 50 ⁇ g poly (I:C) formulated in PBS (200 ⁇ _ total volume) into the lateral flank.
  • C57BL/6 mice were inoculated subcutaneously with lxlO 5 B16F10 melanoma cells into the right flank and randomly distributed into treatment groups. Tumor volume was measured unblinded with a caliper and calculated using the formula (AxB 2 )/2 (A as the largest and B the smallest diameter of the tumor).
  • PBMC peripheral blood cells
  • PBMC peripheral blood cells
  • peptide and syngeneic BMDC peptide and syngeneic BMDC.
  • Subtyping of T cell responses was performed by addition of a MHC class II blocking antibody (20 pg/ml, clone M5/1 14. BioXcell). All samples were tested in duplicates or triplicates.
  • Flow cytometric analysis was used to determine the subtype of mutation reactive T cells.
  • Brefeldin A Sigma-Aldrich
  • 2x10 6 splenocytes w ere stimulated with 2x10 5 RNA transfected BMDC or 2 ⁇ g/mL peptide.
  • PMA phorbol 12-myristatc 13 -acetate
  • lonomycin 1 , ug/ml, Sigma-Aldrich
  • Tumor infiltrating leucocytes were prepared from subcutaneous B16F10 tumors as described previously (PMID:2071934). The resulting cell suspension was stained for CD4, CDS, Gr-1 and CD 1 lb surface marker. Intracellular FoxP3 staining was performed according to the manufacturer ' s protocol (Mouse Foxp3 Buffer Set, BD). Samples were acquired on a BD FACSCanto II.
  • CD3 CD4 + , FoxP3 " and tumor areas were captured on an Axio Scan.Zl (Zeiss) and manually pre-defined tumor and lung regions were quantified via computerized image analysis software (Tissue Studio 3.6.1 , Definiens).
  • Fluorescent labeled antibodies (FoxP3, clone F.IK- 16s, eBioscience; CDS, clone 53-6.7, BD; CD4, clone RM4-5, BD) were diluted in staining buffer (PBS supplemented with 1% BSA, 5% mouse serum and 0.02% Nonident) and sections were stained overnight at 4°C. After washing twice with washing buffer (PBS supplemented with 1% BSA and 0. 02% Nonident) and once with PBS, slides were stained for 3min with Hoechst (Sigma), washed 3 times with PBS, once with distilled water and mounted using Mounting Medium Flouromount G (eBioscience).
  • Immunofluorescence images were acquired using an epifluorescencc microscope (ApoTome, Zeiss). Tumor, CD4, CD8 and FoxP3 stained areas were quantified within manually pre-defined tumor regions via computerized image analysis software (Tissue Studio 3.6.1 Definiens)
  • Example 2 MHC class II restricted T cell epitopes in neo-epltope vaccines
  • A Characterization of T cell subtypes reactive against mutated epitopes
  • GS Fig l a
  • Tumor- bearing C57BL/6 mice were immunized with synthetic 27mer peptides encoding the mutated epitope (mutation in position 14), resulting in T cell responses which conferred in vivo tumor control.
  • mice were vaccinated with synthetic 27m er mutated epitope peptides (Fig lb upper right). Their splenocytes were tested in IFN- ⁇ ELISpot to identify immunogenic mutations for further analysis of subtype and cytokine expression (Fig. l a). About 30% of mutated epitopes were found to induce mutation reactive cytokine secreting T cells in mice (Fig l b). Surprisingly, responses against nearly all mutated epitopes (16/17, 95%) were of CD4 + T cell type (Fig lb, Table 1 ).
  • B 16F10 mutations determined to be immunogenic upon peptide or RNA immunization (as described in Figure 1). (WT, wild type; AA#, number of mutated amino acid; Mut, Mutation)
  • CT26 mutations determined to be immunogenic upon RNA immunization (as described in Figure 1). (WT, wild type; AA#, number of mutated amino acid; Mut, Mutation)
  • RNA as vaccine format.
  • Antigen-encoding synthetic RNA is emerging as promising vaccine technology due to its advantages including its capability to deliver more than one epitope, its selective uptake by antigen presenting cells (APC) and its intrinsic adjuvanticity (Diken, M., et al., Gene Ther 1 8, 702 (201 1); Kreiter, S., et al., Curr Opin Immunol 23, 399 (201 1 ); Pascolo, S., Handb Exp Pharmacol , 221 (2008); Sahin, U., et al., Nat Rev Drug Discov 13, 759 (2014); Van, L. S., et al..
  • RNA stabilizing elements in RNA sequence and liposomal formulation
  • NCR clinical testing
  • RNA manufacturing based on in vitro transcription usually takes a few days ( Figure 3a). At present, the GMP-grade material could be made ready for release within three weeks and this process is continuously being optimized to reduce the duration.
  • RNA pcntatopc was engineered encoding all five mutated epitopes connected by 10m er non-immunogenic glycine/serine linkers to avoid the generation of junctional epitopes (Fig. 3a).
  • Fig. 3a junctional epitopes
  • RNA pentatope vaccines were assessed the anti-tumour efficacy of immune responses elicited b RNA pentatope vaccines in a lung metastasis model of CT26 luciferase transfectant (CT26-Luc) tumors.
  • Tumor-bearing BALB/c mice were vaccinated repeatedly with a mixture of two RNA pentatopes (3 MHC class I and 7 class II restricted epitopes) including the mutations tested in the previous experiment. Tumor growth in vaccinated mice was significantly inhibited as measured by BLI of the lung (Fig 3c left). At day 32 all mice in the RNA pentatope group were alive whereas 80% of the control mice had already died (Fig 3c mid).
  • T cells against each single epitope are elicited with a MERIT approach employing a poly-neo-epitope encoding RNA vaccine. These T cells target tumor lesions, recognize their mutated targets and result in efficient tumor control in vivo.
  • NVRC normalized variant read counts
  • Antigen specific TH cells promote the cross-priming of tumor specific CTL responses by CD40 ligand mediated licensing of dendritic cells. This may result in antigen spread if T H cells recognize their antigen on the same APC that cross-presents an unrelated CTL epitope (Bennett, S. R., et al, Nature 393, 478 ( 1998); Schoenberger, S. P., et al., Nature 393, 480 ( 1998)).
  • MHC class II restricted T cell epitopes are abundant in the cancer mutanome and can be used to customize RNA-based poly-nco-epitope vaccines with substantial therapeutic effect in mouse tumor models.
  • T cell epitopes presented by MHC class II molecules are typically peptides between 8 and 1 1 amino acids in length with well-defined N- and C-termini.
  • MHC class II molecules present longer peptides of 1 -17 amino acids in length with a 9 amino acid MHC II core binding region and variable number of additional flanking amino acids both contributing to the recognition by CD4 + T cells (Arnold, P. Y., et al., J Immunol 169, 739 (2002)).
  • the MERIT approach we presented here integrates advances in the field of next generation sequencing, computational immunology and synthetic genomics and thereby provides the integrated technology for comprehensive exploitation of the neo-epitope target repertoire.
  • Targeting multiple mutations at once may at least in theory pave the way to solve critical problems in current cancer drug development such as clonal heterogeneity and antigen escape (Kroemer, G. and Zitvogel, L., Oncoimmunology 1 , 579 (2012); Mittal, D.. et al., Curr Opin Immunol 27, 16 (2014)).
  • Example 3 Selection of mutations having anti-tumor immunity For selecting/ranking amino acid sequence modifications one may proceed as follows:
  • VAF variant allele frequency
  • o input are short read alignments of an RNA-Scq experiment done with the same tumor sample as used for mutation detection
  • Figure 7 shows the ROC curves for the optimum thresholds, indicating a pronounced influence of the mutation expression for binders with only a mediocre relative binding affinity (Figure 7, right panel, values between a false positive rate of about 0.3 and 0.6).

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PCT/EP2015/053021 WO2016128060A1 (en) 2015-02-12 2015-02-12 Predicting t cell epitopes useful for vaccination
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EP21191307.4A EP3954383A1 (en) 2015-02-12 2016-02-09 Predicting t cell epitopes useful for vaccination
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KR1020177025724A KR102359213B1 (ko) 2015-02-12 2016-02-09 예방접종에 유용한 t 세포 에피토프의 예측 방법
CN201680010134.9A CN107430132B (zh) 2015-02-12 2016-02-09 预测可用于疫苗接种的t细胞表位
JP2017542106A JP6882178B2 (ja) 2015-02-12 2016-02-09 ワクチン接種に有用なｔ細胞エピトープの予測
PT167045673T PT3256853T (pt) 2015-02-12 2016-02-09 Previsão de epítopos de células t úteis para vacinação
HUE16704567A HUE056658T2 (hu) 2015-02-12 2016-02-09 A vakcinázáshoz hasznos T-sejt epitópok prediktálása
ES16704567T ES2888430T3 (es) 2015-02-12 2016-02-09 Predicción de epítopos de células T útiles para la vacunación
CN202110651565.6A CN113484523A (zh) 2015-02-12 2016-02-09 预测可用于疫苗接种的t细胞表位
MX2017010332A MX2017010332A (es) 2015-02-12 2016-02-09 Predicción de epítopos de células t útiles para vacunación.
IL253627A IL253627B (en) 2015-02-12 2017-07-24 Prediction of t-cell epitopes used for vaccination
MX2022000012A MX2022000012A (es) 2015-02-12 2017-08-10 Prediccion de epitopos de celulas t utiles para vacunacion.
MX2023002858A MX2023002858A (es) 2015-02-12 2017-08-10 Prediccion de epitopos de celulas t utiles para vacunacion.
HK18106129.1A HK1246852A1 (zh) 2015-02-12 2018-05-11 預測可用於疫苗接種的t細胞表位
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10055540B2 (en) 2015-12-16 2018-08-21 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
WO2018183980A3 (en) * 2017-03-31 2018-12-20 Pei Jia Yang Ranking system for immunogenic cancer-specific epitopes
CN110741260A (zh) * 2017-06-09 2020-01-31 生物技术Rna制药有限公司 用于预测疾病特异性氨基酸修饰用于免疫治疗的可用性的方法
WO2020118004A1 (en) * 2018-12-05 2020-06-11 Linearx, Inc. Amplicon expression vector vaccines
WO2020132586A1 (en) * 2018-12-21 2020-06-25 Neon Therapeutics, Inc. Method and systems for prediction of hla class ii-specific epitopes and characterization of cd4+ t cells
CN111556760A (zh) * 2017-11-06 2020-08-18 纪念斯隆-凯特林癌症中心 作为疫苗免疫佐剂的热灭活的牛痘病毒
US11156617B2 (en) 2015-02-12 2021-10-26 BioNTech RNA Pharmaceuticals GbmH Predicting T cell epitopes useful for vaccination
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11585805B2 (en) 2016-02-19 2023-02-21 Nantcell, Inc. Methods of immunogenic modulation
US11766473B2 (en) 2016-02-05 2023-09-26 Xeme Biopharma Inc. Therapeutic cancer vaccine containing tumor-associated neoantigens and immunostimulants in a deliver system
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens
US11965892B2 (en) 2017-02-12 2024-04-23 Biontech Us Inc. HLA-based methods and compositions and uses thereof
US12059459B2 (en) 2016-01-08 2024-08-13 Nykode Therapeutics ASA Therapeutic anticancer neoepitope vaccine

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3052106A4 (en) 2013-09-30 2017-07-19 ModernaTX, Inc. Polynucleotides encoding immune modulating polypeptides
BR112016024644A2 (pt) 2014-04-23 2017-10-10 Modernatx Inc vacinas de ácido nucleico
WO2017020026A1 (en) * 2015-07-30 2017-02-02 Modernatx, Inc. Concatemeric peptide epitopes rnas
KR101925040B1 (ko) * 2016-11-11 2018-12-04 한국과학기술정보연구원 Mhc와 펩타이드 사이의 결합 친화성 예측 방법 및 장치
CN110505877A (zh) * 2017-02-01 2019-11-26 摩登纳特斯有限公司 Rna癌症疫苗
MA48047A (fr) 2017-04-05 2020-02-12 Modernatx Inc Réduction ou élimination de réponses immunitaires à des protéines thérapeutiques administrées par voie non intraveineuse, par exemple par voie sous-cutanée
WO2019147921A1 (en) 2018-01-26 2019-08-01 Nantcell, Inc. Rapid verification of virus particle production for a personalized vaccine
CN111836639A (zh) 2018-01-26 2020-10-27 河谷细胞有限公司 用于联合癌症疫苗和免疫辅助疗法的组合物和方法
CN108491689B (zh) * 2018-02-01 2019-07-09 杭州纽安津生物科技有限公司 基于转录组的肿瘤新抗原鉴定方法
US20210388438A1 (en) * 2018-09-27 2021-12-16 Vaccibody As Method for selecting neoepitopes
CA3114265A1 (en) * 2018-11-15 2020-05-22 Nouscom Ag Selection of cancer mutations for generation of a personalized cancer vaccine
CN111621564B (zh) * 2019-02-28 2022-03-25 武汉大学 一种鉴定有效肿瘤新抗原的方法
WO2020221783A1 (en) * 2019-04-29 2020-11-05 Vaccibody As Methods for pre-selection of neoepitopes
KR102184720B1 (ko) * 2019-10-11 2020-11-30 한국과학기술원 암 세포 표면의 mhc-펩타이드 결합도 예측 방법 및 분석 장치
CN111429965B (zh) * 2020-03-19 2023-04-07 西安交通大学 一种基于多连体特征的t细胞受体对应表位预测方法
CN111617238B (zh) * 2020-06-02 2022-12-13 苏州药明康德新药开发有限公司 小鼠ct26结直肠癌治疗性肿瘤多肽疫苗制剂及其制备方法
WO2022013154A1 (en) 2020-07-14 2022-01-20 Myneo Nv Method, system and computer program product for determining presentation likelihoods of neoantigens
WO2022110010A1 (zh) * 2020-11-27 2022-06-02 苏州吉玛基因股份有限公司 免疫激活剂、抗原和抗体在制备预防、治疗肿瘤产品中的应用
CN114984198A (zh) * 2021-02-24 2022-09-02 中国科学院上海药物研究所 包含肿瘤新生抗原纳米制剂和树突状细胞疫苗的试剂盒及其用途
CN113012756A (zh) * 2021-03-08 2021-06-22 杭州纽安津生物科技有限公司 一种个体化肿瘤新生抗原肽的筛选方法及其疫苗制剂
AU2021461416A1 (en) 2021-08-24 2024-02-22 BioNTech SE In vitro transcription technologies
WO2024083345A1 (en) 2022-10-21 2024-04-25 BioNTech SE Methods and uses associated with liquid compositions

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033739A1 (en) 1995-04-25 1996-10-31 Smithkline Beecham Biologicals S.A. Vaccines containing a saponin and a sterol
WO1997024447A1 (en) 1996-01-02 1997-07-10 Chiron Corporation Immunostimulation mediated by gene-modified dendritic cells
WO2012159754A2 (en) * 2011-05-24 2012-11-29 Biontech Ag Individualized vaccines for cancer
WO2014168874A2 (en) * 2013-04-07 2014-10-16 The Broad Institute, Inc. Compositions and methods for personalized neoplasia vaccines
WO2014180569A1 (en) * 2013-05-10 2014-11-13 Biontech Ag Predicting immunogenicity of t cell epitopes

Family Cites Families (104)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5804381A (en) 1996-10-03 1998-09-08 Cornell Research Foundation Isolated nucleic acid molecule encoding an esophageal cancer associated antigen, the antigen itself, and uses thereof
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
US5264618A (en) 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US6235525B1 (en) 1991-05-23 2001-05-22 Ludwig Institute For Cancer Research Isolated nucleic acid molecules coding for tumor rejection antigen precursor MAGE-3 and uses thereof
US7422902B1 (en) 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
DE69723434T2 (de) 1996-04-26 2004-05-19 Rijksuniversiteit Te Leiden Verfahren zur selektion und produktion von t-zell-peptide epitope und vakzine mit diese epitope
ES2187812T3 (es) 1996-09-13 2003-06-16 Lipoxen Technologies Ltd Composicion de liposomas.
EP0839912A1 (en) 1996-10-30 1998-05-06 Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof
US6074645A (en) 1996-11-12 2000-06-13 City Of Hope Immuno-reactive peptide CTL epitopes of human cytomegalovirus
KR20010024585A (ko) 1997-11-06 2001-03-26 로셰 디아그노스틱스 게엠베하 종양-특이적 항원, 이들의 제조방법, 및 이들의 면역화 및진단에서의 용도
US6432925B1 (en) 1998-04-16 2002-08-13 John Wayne Cancer Institute RNA cancer vaccine and methods for its use
EP1117430A1 (en) 1998-10-05 2001-07-25 Genzyme Corporation Genes differentially expressed in cancer cells to design cancer vaccines
EP1178785B1 (en) 1999-05-06 2008-12-24 Wake Forest University Compositions and methods for identifying antigens which elicit an immune response
WO2001047959A2 (en) 1999-11-30 2001-07-05 Ludwig Institute For Cancer Research Isolated nucleic acid molecules encoding cancer associated antigens, the antigens per se, and uses thereof
KR100838507B1 (ko) 1999-12-28 2008-06-17 에피뮨 인코포레이티드 최적화된 미니유전자 및 이에 의해 암호화된 펩타이드
US7462354B2 (en) 1999-12-28 2008-12-09 Pharmexa Inc. Method and system for optimizing minigenes and peptides encoded thereby
JP4776852B2 (ja) * 2000-01-28 2011-09-21 アメリカ合衆国 癌抗原ｎｙｅｓｏ−１由来の新規ｍｈｃクラスｉｉ拘束ｔ細胞エピトープ
WO2001082963A2 (en) * 2000-04-28 2001-11-08 Ctl Immunotherapies Corp. Method of identifying and producing antigen peptides and use thereof as vaccines
EP1292331A2 (en) 2000-06-07 2003-03-19 Biosynexus Incorporated Immunostimulatory rna/dna hybrid molecules
US6472176B2 (en) 2000-12-14 2002-10-29 Genvec, Inc. Polynucleotide encoding chimeric protein and related vector, cell, and method of expression thereof
DK1857122T3 (da) 2001-06-05 2011-03-21 Curevac Gmbh Stabiliseret mRNA med forøget G/C-indhold, kodende for et viralt antigen
DE10162480A1 (de) 2001-12-19 2003-08-07 Ingmar Hoerr Die Applikation von mRNA für den Einsatz als Therapeutikum gegen Tumorerkrankungen
AUPS054702A0 (en) 2002-02-14 2002-03-07 Immunaid Pty Ltd Cancer therapy
DE60328685D1 (de) 2002-06-13 2009-09-17 Merck Patent Gmbh Verfahren für die identifizierung von allo-antigenen und ihren verwendung für krebs therapie und transplantation
DE10229872A1 (de) 2002-07-03 2004-01-29 Curevac Gmbh Immunstimulation durch chemisch modifizierte RNA
JP2006518982A (ja) * 2002-10-02 2006-08-24 エフ．ホフマン−ラ　ロシュ　アーゲー Ｍｈｃｉｉ結合ペプチド
DE10344799A1 (de) 2003-09-26 2005-04-14 Ganymed Pharmaceuticals Ag Identifizierung von Oberflächen-assoziierten Antigenen für die Tumordiagnose und -therapie
JP5645340B2 (ja) 2003-10-15 2014-12-24 メディゲーネ アクチエンゲゼルシャフトＭｅｄｉＧｅｎｅ ＡＧ 活性成分を含有するカチオンリポソームの投与方法
US20070202119A1 (en) 2003-10-24 2007-08-30 Ashdown Martin L Method Of Therapy
US7303881B2 (en) 2004-04-30 2007-12-04 Pds Biotechnology Corporation Antigen delivery compositions and methods of use
DE102004023187A1 (de) 2004-05-11 2005-12-01 Ganymed Pharmaceuticals Ag Identifizierung von Oberflächen-assoziierten Antigenen für die Tumordiagnose und -therapie
DE102004057303A1 (de) 2004-11-26 2006-06-01 Merck Patent Gmbh Stabile Kristallmodifikationen von DOTAP Chlorid
EP1838342A2 (en) 2004-12-29 2007-10-03 Mannkind Corporation Methods to bypass cd+4 cells in the induction of an immune response
EP1715346A1 (en) 2005-04-22 2006-10-25 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Method of identifying CD4+ T cell antigens
NZ563193A (en) 2005-05-09 2010-05-28 Ono Pharmaceutical Co Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
DK1907424T3 (en) 2005-07-01 2015-11-09 Squibb & Sons Llc HUMAN MONOCLONAL ANTIBODIES TO PROGRAMMED death ligand 1 (PD-L1)
LT2578685T (lt) 2005-08-23 2019-06-10 The Trustees Of The University Of Pennsylvania Rnr, apimančios modifikuotus nukleozidus ir jų panaudojimo būdai
DE102005041616B4 (de) 2005-09-01 2011-03-17 Johannes-Gutenberg-Universität Mainz Melanom-assoziierte MHC Klasse I assoziierte Oligopeptide und für diese kodierende Polynukleotide und deren Verwendungen
EP1762575A1 (en) 2005-09-12 2007-03-14 Ganymed Pharmaceuticals AG Identification of tumor-associated antigens for diagnosis and therapy
EP1994181A4 (en) 2006-02-27 2010-05-19 Univ Arizona IDENTIFICATION AND USE OF NOVOPEPTIDES FOR THE TREATMENT OF CANCER
EP2061504A4 (en) 2006-09-20 2010-01-27 Univ Johns Hopkins COMBINATION THERAPY FOR CANCER AND INFECTION DISEASES WITH ANTI-B7-H1 ANTIBODIES
DE102006060824B4 (de) 2006-12-21 2011-06-01 Johannes-Gutenberg-Universität Mainz Nachweis von individuellen T-Zell-Reaktionsmustern gegen Tumor-assoziierte Antigene (TAA) in Tumorpatienten als Basis für die individuelle therapeutische Vakzinierung von Patienten
CN101663323A (zh) 2006-12-27 2010-03-03 埃默里大学 用于治疗传染病和肿瘤的组合物和方法
US8877206B2 (en) 2007-03-22 2014-11-04 Pds Biotechnology Corporation Stimulation of an immune response by cationic lipids
US8140270B2 (en) 2007-03-22 2012-03-20 National Center For Genome Resources Methods and systems for medical sequencing analysis
EP2170959B1 (en) 2007-06-18 2013-10-02 Merck Sharp & Dohme B.V. Antibodies to human programmed death receptor pd-1
EP2060583A1 (en) 2007-10-23 2009-05-20 Ganymed Pharmaceuticals AG Identification of tumor-associated markers for diagnosis and therapy
PT2276486E (pt) 2008-03-24 2013-12-04 4Sc Discovery Gmbh Novas imidazoquinolinas substituídas
CN110075113A (zh) 2008-04-17 2019-08-02 Pds生物科技公司 通过阳离子脂质的对映体刺激免疫应答
DE102008061522A1 (de) 2008-12-10 2010-06-17 Biontech Ag Verwendung von Flt3-Ligand zur Verstärkung von Immunreaktionen bei RNA-Immunisierung
JP5785168B2 (ja) 2009-07-31 2015-09-24 エスリス ゲーエムベーハーｅｔｈｒｉｓ ＧｍｂＨ タンパク質発現用未修飾および修飾ヌクレオチドの組み合わせを有するｒｎａ
WO2013040142A2 (en) 2011-09-16 2013-03-21 Iogenetics, Llc Bioinformatic processes for determination of peptide binding
EP2569633B1 (en) 2010-05-14 2016-02-10 The General Hospital Corporation Compositions and methods of identifying tumor specific neoantigens
HRP20220796T1 (hr) 2010-10-01 2022-10-14 ModernaTX, Inc. Ribonukleinske kiseline koje sadrže n1-metil-pseudouracil i njihove uporabe
CA2831613A1 (en) 2011-03-31 2012-10-04 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
DE102011102734A1 (de) 2011-05-20 2012-11-22 WMF Württembergische Metallwarenfabrik Aktiengesellschaft Vorrichtung zum Aufschäumen von Milch, Getränkebereiter mit dieser Vorrichtung und Verfahren zum Aufschäumen von Milch
WO2012159643A1 (en) 2011-05-24 2012-11-29 Biontech Ag Individualized vaccines for cancer
EP3492109B1 (en) 2011-10-03 2020-03-04 ModernaTX, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
RS63244B1 (sr) 2011-12-16 2022-06-30 Modernatx Inc Kompozicije modifikovane mrna
WO2013106496A1 (en) 2012-01-10 2013-07-18 modeRNA Therapeutics Methods and compositions for targeting agents into and across the blood-brain barrier
WO2013124701A2 (en) 2012-02-20 2013-08-29 Universita' Degli Studi Di Milano New homo- and heterodimeric smac mimetic compounds as apoptosis inducers
US20130255281A1 (en) 2012-03-29 2013-10-03 General Electric Company System and method for cooling electrical components
WO2013151664A1 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of proteins
WO2014012051A1 (en) 2012-07-12 2014-01-16 Persimmune, Inc. Personalized cancer vaccines and adoptive immune cell therapies
RU2019143431A (ru) 2012-11-01 2020-04-28 Фэктор Байосайенс Инк. Способы и продукты для экспрессии белков в клетках
PL2922554T3 (pl) 2012-11-26 2022-06-20 Modernatx, Inc. Na zmodyfikowany na końcach
EP2931319B1 (en) 2012-12-13 2019-08-21 ModernaTX, Inc. Modified nucleic acid molecules and uses thereof
US20160022840A1 (en) 2013-03-09 2016-01-28 Moderna Therapeutics, Inc. Heterologous untranslated regions for mrna
US20160024181A1 (en) 2013-03-13 2016-01-28 Moderna Therapeutics, Inc. Long-lived polynucleotide molecules
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
US20160032316A1 (en) 2013-03-14 2016-02-04 The Trustees Of The University Of Pennsylvania Purification and Purity Assessment of RNA Molecules Synthesized with Modified Nucleosides
US20160032273A1 (en) 2013-03-15 2016-02-04 Moderna Therapeutics, Inc. Characterization of mrna molecules
WO2014144711A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Analysis of mrna heterogeneity and stability
US10590161B2 (en) 2013-03-15 2020-03-17 Modernatx, Inc. Ion exchange purification of mRNA
US11377470B2 (en) 2013-03-15 2022-07-05 Modernatx, Inc. Ribonucleic acid purification
WO2014152030A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Removal of dna fragments in mrna production process
WO2014152027A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Manufacturing methods for production of rna transcripts
WO2015014375A1 (en) 2013-07-30 2015-02-05 Biontech Ag Tumor antigens for determining cancer therapy
US20160194368A1 (en) 2013-09-03 2016-07-07 Moderna Therapeutics, Inc. Circular polynucleotides
CA2923029A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
EP3043826A4 (en) 2013-09-13 2017-05-24 Moderna Therapeutics, Inc. Polynucleotide compositions containing amino acids
WO2015043613A1 (en) 2013-09-26 2015-04-02 Biontech Ag Particles comprising a shell with rna
US20160264614A1 (en) 2013-10-02 2016-09-15 Moderna Therapeutics, Inc. Polynucleotide molecules and uses thereof
WO2015051169A2 (en) 2013-10-02 2015-04-09 Moderna Therapeutics, Inc. Polynucleotide molecules and uses thereof
WO2015058780A1 (en) 2013-10-25 2015-04-30 Biontech Ag Method and kit for determining whether a subject shows an immune response
WO2015085318A2 (en) 2013-12-06 2015-06-11 Moderna Therapeutics, Inc. Targeted adaptive vaccines
US20150167017A1 (en) 2013-12-13 2015-06-18 Moderna Therapeutics, Inc. Alternative nucleic acid molecules and uses thereof
WO2015117620A1 (en) 2014-02-05 2015-08-13 Biontech Ag A cannula, an injection or infusion device and methods of using the cannula or the injection or infusion device
BR112016024644A2 (pt) 2014-04-23 2017-10-10 Modernatx Inc vacinas de ácido nucleico
WO2015172843A1 (en) 2014-05-16 2015-11-19 Biontech Diagnostics Gmbh Methods and kits for the diagnosis of cancer
WO2016062323A1 (en) 2014-10-20 2016-04-28 Biontech Ag Methods and compositions for diagnosis and treatment of cancer
EP3230458B1 (en) 2014-12-12 2020-02-19 CureVac AG Artificial nucleic acid molecules for improved protein expression
AU2015373404B2 (en) 2014-12-30 2021-09-09 CureVac SE Artificial nucleic acid molecules
WO2016128060A1 (en) 2015-02-12 2016-08-18 Biontech Ag Predicting t cell epitopes useful for vaccination
WO2016155809A1 (en) 2015-03-31 2016-10-06 Biontech Rna Pharmaceuticals Gmbh Lipid particle formulations for delivery of rna and water-soluble therapeutically effective compounds to a target cell
EP4407622A2 (en) * 2015-07-14 2024-07-31 Personal Genome Diagnostics Inc. Neoantigen analysis
CN108601731A (zh) 2015-12-16 2018-09-28 磨石肿瘤生物技术公司 新抗原的鉴别、制造及使用
MX2019012433A (es) 2017-04-19 2019-12-11 Gritstone Oncology Inc Identificacion, fabricacion y uso de neoantigenos.
AU2018279627B2 (en) 2017-06-09 2023-08-10 Gritstone Bio, Inc. Neoantigen identification, manufacture, and use
US20200363414A1 (en) 2017-09-05 2020-11-19 Gritstone Oncology, Inc. Neoantigen Identification for T-Cell Therapy
CA3078744A1 (en) 2017-10-10 2019-04-18 Gritstone Oncology, Inc. Neoantigen identification using hotspots
CA3083097A1 (en) 2017-11-22 2019-05-31 Gritstone Oncology, Inc. Reducing junction epitope presentation for neoantigens
US20200411135A1 (en) 2018-02-27 2020-12-31 Gritstone Oncology, Inc. Neoantigen Identification with Pan-Allele Models

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033739A1 (en) 1995-04-25 1996-10-31 Smithkline Beecham Biologicals S.A. Vaccines containing a saponin and a sterol
WO1997024447A1 (en) 1996-01-02 1997-07-10 Chiron Corporation Immunostimulation mediated by gene-modified dendritic cells
WO2012159754A2 (en) * 2011-05-24 2012-11-29 Biontech Ag Individualized vaccines for cancer
WO2014168874A2 (en) * 2013-04-07 2014-10-16 The Broad Institute, Inc. Compositions and methods for personalized neoplasia vaccines
WO2014180569A1 (en) * 2013-05-10 2014-11-13 Biontech Ag Predicting immunogenicity of t cell epitopes

Non-Patent Citations (67)

* Cited by examiner, † Cited by third party
Title
ABASTADO ET AL., J. 1MMUNOL, vol. 151, no. 7, January 1999 (1999-01-01), pages 3569 - 75
ALEXANDROV, L. B. ET AL., NATURE, vol. 500, 2013, pages 415
ALI MORTAZAVI ET AL., NATURE METHODS, vol. 5, 2008, pages 621 - 628
ALLARD ET AL., CLIN CANCER RES, vol. 10, 2004, pages 6897 - 6904
ARNOLD, P. Y. ET AL., J IMMUNOL, vol. 169, 2002, pages 739
BENNETT, S. R. ET AL., NATURE, vol. 393, 1998, pages 478
BRITTEN, C. M. ET AL., NAT BIOTECHNOL, vol. 31, 2013, pages 880
CASTLE, J. C. ET AL., BMC GENOMICS, vol. 15, 2014, pages 190
CASTLE, J. C. ET AL., CANCER RES, vol. 72, 2012, pages 1081
CASTLE, J. C. ET AL., SCI REP, vol. 4, 2014, pages 4743
CHOI ET AL., PROC. NATL. ACAD. SCI USA, vol. 106, 2009, pages 19096 - 19101
CROXFORD, J. L. ET AL., AUTOIMMUN REV, vol. 1, 2002, pages 251
D. A. WICK ET AL: "Surveillance of the Tumor Mutanome by T Cells during Progression from Primary to Recurrent Ovarian Cancer", CLINICAL CANCER RESEARCH, vol. 20, no. 5, 2013, US, pages 1125 - 1134, XP055231808 *
DIKEN, M. ET AL., GENE THER, vol. 18, 2011, pages 702
DUAN, F. ET AL., J EXP MED, vol. 211, 2014, pages 2231
DUBEY, P. ET AL., J EXP MED, vol. 185, 1997, pages 695
FAWCETT T., PATTERN RECOGN LETT., vol. 27, 2006, pages 861 - 874
GERLINGER, M. ET AL., N ENGL J MED, vol. 366, 2012, pages 883
GRISWOLD, D. P.; CORBETT, T. H., CANCER, vol. 36, 1975, pages 2441
H. G. W. LEUENBERGER, B. NAGEL, ET AL.,: ""A multilingual glossary of biotechnological terms: (IUPAC Recommendations)",", 1995
HODGES ET AL., NAT. GENET., vol. 39, 2007, pages 1522 - 1527
HOLTKAMP, S. ET AL., BLOOD, vol. 108, 2006, pages 4009
HOYT ET AL., EMBO J., vol. 25, no. 8, 2006, pages 1720 - 9
J. C. CASTLE ET AL: "Exploiting the Mutanome for Tumor Vaccination", CANCER RESEARCH, vol. 72, no. 5, 2012, US, pages 1081 - 1091, XP055231746 *
J. SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 2nd Edition,", 1989, COLD SPRING HARBOR LABORATORY PRESS
KOEBEL, C. M. ET AL., NATURE, vol. 450, 2007, pages 903
KREITER, S. ET AL., CANCER RES, vol. 70, 2010, pages 9031
KREITER, S. ET AL., CURR OPIN IMMUNOL, vol. 23, 2011, pages 399
KREITER, S. ET AL., J IMMUNOL, vol. 180, 2008, pages 309
KRIEG ET AL., NATURE, vol. 374, 1995, pages 546 - 549
KROEMER, G.; ZITVOGEL, L., ONCOIMMUNOLOGY, vol. 1, 2012, pages 579
KUHN, A. N. ET AL., GENE THER, vol. 17, 2010, pages 961
LENNERZ, V. ET AL., PROC NATL ACAD SCI U S A, vol. 102, 2005, pages 16013
LOWER, M. ET AL., PLOS COMPUT BIOL, vol. 8, 2012, pages EL002714
LU, Y. C. ET AL., J IMMUNOL, vol. 190, 2013, pages 6034
MAHVI ET AL., IMMUNOLOGY AND CELL BIOLOGY, vol. 75, 1997, pages 456 - 460
MALOY ET AL., PROC NATL ACAD SCI USA, vol. 98, 2001, pages 3299 - 303
MATSUSHITA, H. ET AL., NATURE, vol. 482, 2012, pages 400
MITTAL, D. ET AL., CURR OPIN IMMUNOL, vol. 27, 2014, pages 16
NAGRATH ET AL., NATURE, vol. 450, 2007, pages 1235 - 1239
NG, PC ET AL., PLOS GEN., vol. 4, no. 8, 2008, pages 1 - 15
PASCOLO, S.: "Handb Exp Pharmacol", vol. 221, 2008
ROBBINS, P. F. ET AL., NAT MED, vol. 19, 2013, pages 747
RONAGHI ET AL.: "A sequencing method based on real-time pyrophosphate", SCIENCE, vol. 281, no. 5375, 1998, pages 363 - 365
S KREITER ET AL: "Mutant MHC class II epitopes drive therapeutic immune responses to cancer", NATURE, vol. 520, no. 7549, 22 April 2015 (2015-04-22), United Kingdom, pages 692 - 696, XP055231810 *
SAHIN, U. ET AL., NAT REV DRUG DISCOV, vol. 13, 2014, pages 759
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 2nd Edition", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHLESSINGER, PROTEINS, vol. 61, no. 1, 2005, pages 115 - 26
SCHOENBERGER, S. P. ET AL., NATURE, vol. 393, 1998, pages 480
SCHUMACHER, T. ET AL., NATURE, vol. 512, 2014, pages 324
SCHUMACHER. T. ET AL., NATURE, vol. 512, 2014, pages 324
SCIENCE, vol. 268, 1995, pages 1432 - 1434
SHAUNA CULSHAW ET AL: "Assessment of Human Immune Response to Mutans Streptococcal Glucosyltransferase Peptides Selected by MHC Class II Binding Probability", INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS, vol. 13, no. 4, 2007, pages 525 - 531, XP019554562 *
SO ET AL., MOL. CELLS, vol. 7, 1997, pages 178 - 186
TEER; MULLIKIN, HUMAN MOL GENET, vol. 19, no. 2, 2010, pages R145 - 51
TRAN, E. ET AL., SCIENCE, vol. 344, 2014, pages 641
V BOISGUÉRIN ET AL: "Translation of genomics-guided RNA-based personalised cancer vaccines: towards the bedside", BRITISH JOURNAL OF CANCER, vol. 111, no. 8, 2014, pages 1469 - 1475, XP055231809 *
VAN, L. S. ET AL., HUM VACCIN IMMUNOTHER, vol. 9, 2013
VAN, R. N. ET AL., J CLIN ONCOL, vol. 31, 2013, pages E439 - E442
VITA, R. ET AL., NUCLEIC ACIDS RES, vol. 38, 2010, pages D854 - D862
VOELKERDING ET AL.: "Next generation sequencing: From basic research to diagnostics", CLINICAL CHEMISTRY, vol. 55, 2009, pages 641 - 658
WANG P ET AL., BMC BIOINFORMATICS, vol. 11, 2010, pages 568, Retrieved from the Internet <URL:http://tools.immuneepitope.org/mhcii>
WOLFEL, T. ET AL., SCIENCE, vol. 269, 1995, pages 1281
WW OVERWIJK ET AL: "Mining the mutanome: developing highly personalized Immunotherapies based on mutational analysis of tumors", JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 1, no. 1, 2013, pages 11, XP021157683 *
ZHANG ET AL., J. BIOL. CHEM., vol. 279, no. 10, 2004, pages 8635 - 41
ZHANG ET AL.: "The impact of next-generation sequencing on genomics", J GENET GENOMICS, vol. 38, no. 3, 2011, pages 95 - 109
ZHU S ET AL: "Improving MHC binding peptide prediction by incorporating binding data of auxiliary MHC molecules", BIOINFORMATICS, vol. 22, no. 13, 2006, pages 1648 - 1655, XP002690592 *

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* Cited by examiner, † Cited by third party
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US10847252B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US10847253B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US12059459B2 (en) 2016-01-08 2024-08-13 Nykode Therapeutics ASA Therapeutic anticancer neoepitope vaccine
US11766473B2 (en) 2016-02-05 2023-09-26 Xeme Biopharma Inc. Therapeutic cancer vaccine containing tumor-associated neoantigens and immunostimulants in a deliver system
US11585805B2 (en) 2016-02-19 2023-02-21 Nantcell, Inc. Methods of immunogenic modulation
US11965892B2 (en) 2017-02-12 2024-04-23 Biontech Us Inc. HLA-based methods and compositions and uses thereof
WO2018183980A3 (en) * 2017-03-31 2018-12-20 Pei Jia Yang Ranking system for immunogenic cancer-specific epitopes
US11485784B2 (en) 2017-03-31 2022-11-01 Act Genomics (Ip) Co., Ltd. Ranking system for immunogenic cancer-specific epitopes
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